Rna interference for the treatment of gain-of-function disorders

ABSTRACT

The present invention relates to the discovery of an effective treatment for a variety of gain-of-function diseases, in particular, Huntington&#39;s disease (HD). The present invention utilizes RNA Interference technology (RNAi) against polymorphic regions in the genes encoding various gain-of-function mutant proteins resulting in an effective treatment for the gain-of-function disease.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/571,705, filed Dec. 9, 2008, entitled “RNA Interference forthe Treatment of Gain-of-Function Disorders,” which is a national stageapplication of PCT Application No. PCT/US2004/029968, filed Sep. 13,2004, which claims the benefit of priority to U.S. Provisional PatentApplication No. 60/502,678, filed Sep. 12, 2003. This application is acontinuation-in-part of U.S. patent application Ser. No. 12/348,794,filed Jan. 5, 2009, entitled “RNA Silencing Compositions and Methods forthe Treatment of Huntington's Disease,” which is a continuation of PCTApplication No. PCT/US2007/015638, filed Jul. 9, 2007, which claims thebenefit of priority to U.S. Provisional Patent Application No.60/819,704, filed Jul. 7, 2006. The entire contents of the foregoingpatent applications are incorporated herein by reference.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant No.NS038194, awarded by the National Institutions of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is the mechanism of sequence-specific,post-transcriptional gene silencing initiated by double-stranded RNAs(dsRNA) homologous to the gene being suppressed. dsRNAs are processed byDicer, a cellular ribonuclease III, to generate duplexes of about 21 ntwith 3′-overhangs (small interfering RNA, siRNA) which mediatesequence-specific mRNA degradation. In mammalian cells siRNA moleculesare capable of specifically silencing gene expression without inductionof the unspecific interferon response pathway. Thus, siRNAs have becomea new and powerful alternative to other genetic tools such as antisenseoligonucleotides and ribozymes to analyze gene function. Moreover,siRNA's are being developed for therapeutic purposes with the aim ofsilencing disease genes in humans.

RNA silencing refers to a group of sequence-specific regulatorymechanisms (e.g. RNA interference (RNAi), transcriptional gene silencing(TGS), post-transcriptional gene silencing (PTGS), quelling,co-suppression, and translational repression) mediated by RNA silencingagents which result in repression or “silencing” of a correspondingprotein-coding gene. RNA silencing has been observed in many types ofeurkayotes, including humans, and utility of RNA silencing agents asboth therapeutics and research tools is the subject of intense interest.

Several types of small (˜19-23 nt), noncoding RNAs trigger RNA silencingin eukaryotes, including small interfering RNAs (siRNAs) and microRNAs(miRNAs, also known as small temporal RNAs (stRNAs)). Recent evidencesuggests that the two classes of small RNAs are functionallyinterchangeable, with the choice of RNA silencing mechanism (e.g.RNAi-mediated mRNA cleavage or translational repression) determinedlargely by the degree of complementarity between the small RNA and itstarget (Schwarz and Zamore, 2002; Hutvagner and Zamore, 2002; Zeng etal., 2003; Doench et al., 2003). RNA silencing agents with a high degreeof complementarity to a corresponding target mRNA have been shown todirect its silencing by the cleavage-based mechanism (Zamore et al.,2000; Elbashir et al., 2001a; Rhoades et al., 2002; Reinhart et al.,2002; Llave et al., 2002a; Llave et al., 2002b; Xie et al., 2003;Kasschau et al., 2003; Tang et al., 2003; Chen, 2003). RNA silencingagents with a lower degree of complementarity mediate gene silencing byrecruiting the RISC complex to the target mRNA, thereby blocking itstranslation but leaving the mRNA intact (Mourelatos et al., 2002;Hutvagner and Zamore, 2002; Caudy et al., 2002; Martinez et al., 2002;Abrahante et al., 2003; Brennecke et al., 2003; Lin et al., 2003; Xu etal., 2003).

RNA silencing agents have received particular interest as research toolsand therapeutic agents for their ability to knock down expression of aparticular protein with a high degree of sequence specificity. Thesequence specificity of RNA silencing agents is particularly useful forallele-specific silencing dominant, gain-of-function gene mutations.Diseases caused by dominant, gain-of-function gene mutations develop inheterozygotes bearing one mutant and one wild type copy of the gene.Some of the best-known diseases of this class are commonneurodegenerative diseases, including Alzheimer's disease, Parkinson'sdisease and amyotrophic lateral sclerosis (ALS; “Lou Gehrig's disease”)(Taylor et al., 2002). In these diseases, the exact pathways whereby themutant proteins cause cell degeneration are not clear, but the origin ofthe cellular toxicity is known to be the mutant protein.

One group of inherited gain-of-function disorders are known as thetrinucleotide repeat diseases. The common genetic mutation among thesediseases is an increase in a series of a particular trinucleotiderepeat. To date, the most frequent trinucleotide repeat is CAG, whichcodes for the amino acid glutamine. At least 9 CAG repeat diseases areknown and there are more than 20 varieties of these diseases, includingHuntington's disease, Kennedy's disease and many spinocerebellardiseases. These disorders share a neurodegenerative component in thebrain and/or spinal cord. Each disease has a specific pattern ofneurodegeneration in the brain and most have an autosomal dominantinheritance. The onset of the diseases generally occurs at 30 to 40years of age, but in Huntington's disease CAG repeats in the huntingtingene of >60 portend a juvenile onset.

Recent research by the instant inventors has shown that the geneticmutation (increase in length of CAG repeats from normal <36 in thehuntingtin gene to >36 in disease) is associated with the synthesis of amutant huntingtin protein, which has >36 polyglutamines (Aronin et al.,1995). It has also been shown that the protein forms cytoplasmicaggregates and nuclear inclusions (Difiglia et al., 1997) and associateswith vesicles (Aronin et al., 1999). The precise pathogenic pathways arenot known.

Huntington's disease (and by implication other trinucleotide repeatdiseases) is believed to be caused, at least in part, by aberrantprotein interactions, which cause impairment of critical neuronalprocesses, neuronal dysfunction and ultimately neuronal death(neurodegeneration in brain areas called the striatum and cortex).

In the search for an effective treatment for these diseases, researchersin this field emphasized understanding the pathogenesis of the diseaseand initially sought to intercede at the level of the presumed aberrantprotein interactions. However, there is no approved treatment forHuntington's disease or other trinucleotide repeat diseases.Accordingly, therapeutic RNA silencing agents capable of silencingHuntingtin proteins are of considerable interest.

SUMMARY OF THE INVENTION

The present invention relates to the methods for treating a variety ofgain-of-function diseases. In particular, the invention provides methodsfor the selective destruction of mutant mRNAs transcribed fromgain-of-function mutant genes, thus preventing production of the mutantproteins encoded by such genes. Other RNAi-based methods for destroyingmutant genes have been proposed in which siRNAs are targeted to, forexample, a point mutation occurring in a single allele in the mutantgene (e.g., the point mutation in the superoxide dismutase (SOD) geneassociated with amyotrophic lateral sclerosis (ALS)). However, there isa key difference between ALS and trinucleotide repeat diseases, such asHuntington's disease. ALS has a point mutation in one allele as thegenetic change whereas trinucleotide repeat diseases have an expandedCAG repeat region in one allele as the genetic change. Use of RNAiagainst the expanded CAG repeat region has potential complications. Over80 normal genes with CAG repeat regions are known to exist in cells.Thus, siRNAs targeting these CAG repeats cannot be used without riskingwidespread destruction of normal CAG repeat-containing mRNAs. Likewise,targeting non-allele-specific sites would result in loss of both normaland mutant huntingtin causes neuronal dysfunction.

The methods of the invention utilize RNA interference technology (RNAi)against selected polymorphic regions (i.e., regions containingallele-specific or allelic polymorphisms) which are distinct from thesite of mutation in the genes encoding mutant proteins. Themethodologies of the instant invention are effective treatments forgain-of-function diseases resulting from deletion mutations, insertionmutations, point mutations, and the like, provided that the mutant geneencodes a protein having a function not normally associated with wildtype protein.

In a preferred aspect, the methodologies of the instant inventionprovide an effective treatment for Huntington's disease (HD). Themethodologies also provide effective treatments for other polyglutaminedisorders and/or trinucleotide repeat disease, as described in detailherein.

Accordingly, in one aspect, the present invention provides a method oftreating a subject having or at risk of having a disease characterizedor caused by a gain of function mutant protein by administering to thesubject an effective amount of an RNAi agent targeting an allelicpolymorphism within a gene encoding a mutant protein e.g.,) huntingtinprotein, such that sequence-specific interference of a gene occursresulting in an effective treatment for the disease. In one embodiment,the mutant protein contains an expanded polyglutamine region. In anotherone embodiment, the gene encoding the mutant protein contains anexpanded trinucleotide repeat region.

In a yet another embodiment, the method of the invention can be used totreat Huntington's disease and a variety of other diseases selected fromthe group consisting of spino-cerebellar ataxia type 1, spino-cerebellarataxia type 2, spino-cerebellar ataxia type 3, spino-cerebellar ataxiatype 6, spino-cerebellar ataxia type 7, spino-cerebellar ataxia type 8,spino-cerebellar ataxia type 12, myotonic dystrophy, spinal bulbarmuscular disease and dentatoiubral-pallidoluysian atrophy.

The method of the invention uses RNAi agents homologous to an allelicpolymorphism within the gene encoding, for example, a mutant huntingtinprotein for the treatment of Huntington's disease. In a preferredembodiment, the RNAi agent targets allelic polymorphism selected fromthe group consisting of P1-P5. In a further preferred embodiment, theRNAi agent targets an allelic polymorphism selected from the groupconsisting of P6-P43.

In a further embodiment, the invention provides RNAi agents comprisingof a first and second strand each containing 16-25 nucleotides. Thefirst strand of the present invention is homologous to a region of agene encoding a gain-of-function mutant protein, wherein the nucleotidesequence of the gain-of-function mutant protein comprises an allelicpolymorphism. The second strand includes 16-25 nucleotides complementaryto the first strand. The RNAi agent can also have a loop portioncomprising 4-11, e.g., 4, 5, 6, 7, 8, 9, 10, 11, nucleotides thatconnects the two nucleotides sequences. In still other embodiments, thetarget region of the mRNA sequence is located in a 5′ untranslatedregion (UTR) or a 3′ UTR of the mRNA of a mutant protein.

In another embodiment, the invention provides an expression constructcomprising an isolated nucleic acid that encodes a nucleic acid moleculewith a first sequence of 16-25 nucleotides homologous to an allelicpolymorphism within, for example, the gene encoding a mutant huntingtinprotein. The expression construct can be for example, a viral vector,retroviral vector, expression cassette or plasmid. The expressionconstruct can also have an RNA polymerase II promoter sequence or RNAPolymerase II promoter sequence, such as, U6 snRNA promoter of H1promoter.

In yet other embodiments, the present invention provides host cellse.g.,) mammalian cells) comprising nucleic acid molecules and expressionconstructs of the present invention.

In still other embodiments, the present invention provides therapeuticcompositions comprising the nucleic acid molecules of the invention anda pharmaceutically acceptable carrier.

In other aspects, the present invention is based, at least in part, onthe discovery of single nucleotide polymorphism (SNP) sites in theHuntingtin (htt) gene which are preferred target sites for RNAsilencing. The htt SNP sites of the invention are relatively prevalentwithin a sample population. In particular, the htt SNPs of the inventionare present within a population at an allelic frequency of at least 30%.Such SNPs sites may be analysed in a patient to determine if they areheterozygous. Each SNP allele of a heterozygous SNP site may then besequenced in the patient to determine which SNP allele is linked withthe expanded CAG repeat region of the HD-associated allele to form aHD-associated haplotype. Such HD-associated htt SNPs are attractivetargets for therapeutic RNA silencing agents and circumventcomplications associated with directly targeting the expanded CAG repeatregion of htt. Over 80 normal genes with CAG repeat regions are known toexist in cells. Thus, RNA silencing agents targeting these CAG repeatscannot be used without risking widespread destruction of normal CAGrepeat-containing mRNAs. Likewise, targeting non-allele-specific siteswould result in loss of both normal and mutant huntingtin causesneuronal dysfunction.

In one aspect, the invention is directed to a method of treating asubject having or at risk for Huntington's disease, comprising:administering to said subject an effective amount of a RNA silencingagent targeting a heterozygous single nucleotide polymorphism (SNP)within a target mRNA encoding a mutant huntingtin (htt) protein, suchthat RNA silencing of said mRNA occurs; thereby treating said disease insaid subject, wherein the SNP has an allelic frequency of at least 10%(e.g., at least 15%, 20%, 25%, 30%, 35%, 40% or more) in a samplepopulation.

In another aspect, the invention is directed to a method of silencing atarget mRNA encoding a mutant huntingtin (htt) protein in a cell,comprising contacting the cell with effective amount of a RNA silencingagent targeting a heterozygous single nucleotide polymorphism (SNP)within the target mRNA, such that RNA silencing of said mRNA occurs,wherein the SNP has an allelic frequency of at least 10% (e.g., at least15%, 20%, 25%, 30%, 35%, 40% or more) in a sample population.

In another aspect, the invention is directed to an RNA silencing agentcomprising an antisense strand comprising about 16-25 nucleotideshomologous to a region of an mRNA encoding a mutant huntingtin (htt)protein, said region comprising a heterozygous single nucleotidepolymorphism (SNP) allele having an allelic frequency of at least 10%(e.g., at least 15%, 20%, 25%, 30%, 35%, 40% or more) in a samplepopulation, wherein the RNA silencing agent is capable of directing RNAsilencing of said mRNA. In certain embodiments, the heterozygous SNPallele is found at a SNP site selected from the group consisting ofRS362331, RS4690077, RS363125, 47 bp into Exon 25, RS363075, RS362268,RS362267, RS362307, RS362306, RS362305, RS362304, and RS362303.

In one embodiment, the SNP allele is present at SNP target siteRS363125. In a particular embodiment, the SNP allele is a C nucleotide.In another particular embodiment, the SNP allele is a U nucleotide.

In another embodiment, the SNP allele is present at SNP target siteRS362331. In a particular embodiment, the SNP allele is an A nucleotide.In another particular embodiment, the SNP allele is a C nucleotide.

In one embodiment, the target mRNA comprises the sequence set forth asSEQ ID NO: 36. In another embodiment, the target mRNA comprises thesequence set forth as SEQ ID NO: 37. In another embodiment, the targetmRNA comprises the sequence set forth as SEQ ID NO: 42. In anotherembodiment, the target mRNA comprises the sequence set forth as SEQ IDNO:43. In another embodiment, the target mRNA comprises the sequence setforth as SEQ ID NO:48.

In certain embodiments, the RNA silencing agent is capable of inducingdiscriminatory RNA silencing.

In one embodiment, the antisense strand of said RNA silencing agent iscomplementary to the SNP and wherein said RNA silencing agent is capableof substantially silencing the mutant huntingtin protein withoutsubstantially silencing the corresponding wild-type huntingtin protein.

In certain preferred embodiments, the RNA silencing agent is an siRNA.In one embodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO: 34; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO: 35. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO: 38; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO: 39. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO:40; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO:41. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO:44; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO:45. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO:46; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO:47. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO:49; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO:50. In anotherembodiment, at least one nucleotide of the siRNA is modified with anucleotide analog or backbone modification (e.g., a phosphorothioate orLocked Nucleic Acid (LNA) modification) which confers, for example,enhanced nuclease resistance.

In other embodiments, the siRNA comprises a lipophilic moiety. In aparticular preferred embodiment, the lipophilic moiety is a cholesterolmoiety.

In other embodiments, the siRNA is an asymmetric siRNA.

In certain embodiments, the subject is identified as having said SNP by(i) providing DNA from the subject; and (ii) sequencing the huntingtingene, or portion thereof, using said DNA. In other embodiments, thesample population is of Western European origin.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a-k: Human huntingtin gene, nucleotide sequence (SEQ ID NO:1)

FIG. 2 a-b: Human huntingtin protein, amino acid sequence (SEQ ID NO:2)

FIG. 3: Sense (SEQ ID NO: 3) and antisense (SEQ ID NO: 4) of thehuntingtin (htt) target RNA sequence

FIG. 4: Thermodynamic analysis of siRNA strand 5′ ends for the siRNAduplex

FIG. 5 a-c: In vitro RNAi reactions programmed with siRNA targeting apolymorphism within the huntingtin (htt) mRNA. (a) Standard siRNA. (b)siRNA improved by reducing the base-pairing strength of the 5′ end ofthe anti-sense strand of the siRNA duplex. (c) siRNA improved byreducing the unpairing the 5′ end of the anti-sense strand of the siRNAduplex.

FIG. 6 a-b. RNAi of endogenous Htt protein in HeLa cells. (a) Immunoblotof human Htt protein. (b) Quantification of same.

FIG. 7: Frequency of SNP heterozygosity at SNP sites located in thehuman Huntingtin gene. The identity of the SNP at each SNP site in thetarget gene and target mRNA are also indicated.

FIG. 8 a-b: In vitro RNAi reactions programmed with siRNA targeting afirst SNP allele (C) in the heterozygous SNP site RS363331 within thehuman huntingtin gene. (a) Sequence of the siRNA (SEQ ID NO: 34; sensestrand; SEQ ID NO: 35, guide strand), which is fully complementary tothe target hht mRNA containing the “C” SNP allele (SEQ ID NO: 36) butwhich forms a G:U mismatch at position 10 (P10) with the non-target mRNAencoded by the corresponding “U” SNP (SEQ ID NO: 37). (b) DiscriminatoryRNA silencing (expressed as units of luciferase reporter gene activityrelative to GFP) by the siRNA in (a) for the targeted “U” SNP allele(“match”) versus the non-targeted “C” SNP allele (“mismatch”).

FIG. 9 a-b: In vitro RNAi reactions programmed with siRNA targeting asecond SNP allele (T) at heterozygous SNP site RS363331 within the humanhuntingtin gene. (a) Sequence of the siRNA (SEQ ID NO: 38; sense strand;SEQ ID NO: 39, guide strand) which is fully complementary to the targethht mRNA containing the “U” SNP allele (SEQ ID NO: 37) but which forms aA:C mismatch at position 10 (P10) with the non-target mRNA encoded bythe corresponding “C” SNP (SEQ ID NO: 36). (b) Discriminatory RNAsilencing (expressed as units of luciferase reporter gene activityrelative to GFP) by the siRNA in (a) for the targeted “U” SNP allele(“match”) versus the non-targeted “C” SNP allele (“mismatch”).

FIG. 10 a-b: In vitro RNAi reactions programmed with siRNA targeting afirst SNP allele (A) in the heterozygous SNP site RS363125 within thehuman huntingtin gene. (a) Sequence of the siRNA (SEQ ID NO: 40; sensestrand; SEQ ID NO: 41, guide strand), which is fully complementary tothe target hht mRNA containing the “A” SNP allele (SEQ ID NO: 42) butwhich forms a U:C mismatch at position 10 (P10) with the non-target mRNAencoded by the corresponding “C” SNP (SEQ ID NO: 43). (b) DiscriminatoryRNA silencing (expressed as units of luciferase reporter gene activityrelative to GFP) by the siRNA in (a) for the targeted “A” SNP allele(“match”) versus the non-targeted “C” SNP allele (“mismatch”).

FIG. 11 a-b: In vitro RNAi reactions programmed with siRNA targeting thesecond SNP allele (“C”) at the heterozygous SNP site RS363125 within thehuman huntingtin gene. (a) Sequence of the siRNA (SEQ ID NO: 44; sensestrand; SEQ ID NO: 45, guide strand) which is fully complementary to thetarget hht mRNA containing the “C” SNP allele (SEQ ID NO: 43) but whichforms a G:A mismatch at position 10 (P10) with the non-target mRNAencoded by the corresponding “C” SNP (SEQ ID NO: 42). (b) DiscriminatoryRNA silencing (expressed as units of luciferase reporter gene activityrelative to GFP) by the siRNA in (a) for the targeted “C” SNP allele(“match”) versus the non-targeted “A” SNP allele (“mismatch”).

FIG. 12 a-d: In vitro RNAi reactions performed in HEK cells homozygousfor the C polymorphism in the 3′ UTR of the human huntingtin (htt) gene.(a) Sequence of a matched siRNA (SEQ ID NO:46, sense strand; SEQ IDNO:47, guide strand) having a guide strand that is perfectlycomplementary to the target site in the homozygous target allele (SEQ IDNO:48). (b) Sequence of a mismatched siRNA (SEQ ID NO:49, sense strand;SEQ ID NO:50, guide strand) having a guide strand which forms a U:Cmismatch at position 10 (P10) with the homozygous target allele. (c)Relative change in htt target mRNA levels in HEK cells transfected with20 nM of siRNAs depicted in (a) and (b) versus mock transfection asmeasured by quantitative RT-PCR. (d) Relative change in Htt proteinlevels in HEK cells transfected with 20 nM of siRNAs depicted in (a) and(b) versus mock transfection as measured by Western blot.

FIG. 13: Relative change in htt target mRNA levels in HEK cellstransfected with 5, 10, or 20 nM of the matched and mismatched siRNAsdepicted in FIGS. 12( a) and 12(b) and an unrelated GFP siRNA asmeasured by quantitative RT-PCR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and reagents for treating avariety of gain-of-function diseases. In one aspect, the inventionrelates to methods and reagents for treating a variety of diseasescharacterized by a mutation in one allele or copy of a gene, themutation encoding a protein which is sufficient to contribute to orcause the disease. Preferably, the methods and reagents are used totreat diseases caused or characterized by a mutation that is inheritedin an autosomal dominant fashion. In one embodiment, the methods andreagents are used for treating a variety of neurodegenerative diseasecaused by a gain-of-function mutation, e.g., polyglutamine disordersand/or trinucleotide repeat diseases, for example, Huntington's disease.In another embodiment, the methods and reagents are used for treatingdiseases caused by a gain-of-function in an oncogene, the mutated geneproduct being a gain-of-function mutant, e.g., cancers caused by amutation in the ret oncogene (e.g., ret-1), for example, endocrinetumors, medullary thyroid tumors, parathyroid hormone tumors, multipleendocrine neoplasia type2, and the like. In another embodiment, themethods and reagents of the invention can be used to treat a variety ofgastrointestinal cancers known to be caused by an autosomally-inherited,gain-of-function mutations.

The present invention utilizes RNA interference technology (RNAi)against allelic polymorphisms located within a gene encoding again-of-function mutant protein. RNAi destroys the corresponding mutantmRNA with nucleotide specificity and selectivity. RNA agents of thepresent invention are targeted to polymorphic regions of a mutant gene,resulting in cleavage of mutant mRNA. These RNA agents, through a seriesof protein-nucleotide interactions, function to cleave the mutant mRNAs.Cells destroy the cleaved mRNA, thus preventing synthesis ofcorresponding mutant protein e.g., the huntingtin protein.

Accordingly, in one aspect, the present invention provides a method oftreating a subject having or at risk of having a disease characterizedor caused by a gain of function mutant protein by administering to thesubject an effective amount of an RNAi agent targeting an allelicpolymorphism within a gene encoding a mutant protein e.g.,) huntingtinprotein, such that sequence-specific interference of a gene occursresulting in an effective treatment for the disease. In one embodiment,the mutant protein contains an expanded polyglutamine region. In anotherone embodiment, the gene encoding the mutant protein contains anexpanded trinucleotide repeat region.

In a yet another embodiment, the method of the invention can be used totreat Huntington's disease and a variety of other diseases selected fromthe group consisting of spino-cerebellar ataxia type 1, spino-cerebellarataxia type 2, spino-cerebellar ataxia type 3, spino-cerebellar ataxiatype 6, spino-cerebellar ataxia type 7, spino-cerebellar ataxia type 8,spino-cerebellar ataxia type 12, myotonic dystrophy, spinal bulbarmuscular disease and dentatoiubral-pallidoluysian atrophy.

The method of the invention uses RNAi agents homologous to an allelicpolymorphism within the gene encoding, for example, a mutant huntingtinprotein for the treatment of Huntington's disease. In a preferredembodiment, the RNAi agent targets allelic polymorphism selected fromthe group consisting of P1-P5. In a further preferred embodiment, theRNAi agent targets an allelic polymorphism selected from the groupconsisting of P6-P43.

In a further embodiment, the invention provides RNAi agents comprisingof a first and second strand each containing 16-25 nucleotides. Thefirst strand of the present invention is homologous to a region of agene encoding a gain-of-function mutant protein, wherein the nucleotidesequence of the gain-of-function mutant protein comprises an allelicpolymorphism. The second strand includes 16-25 nucleotides complementaryto the first strand. The RNAi agent can also have a loop portioncomprising 4-11, e.g., 4, 5, 6, 7, 8, 9, 10, 11, nucleotides thatconnect the two nucleotides sequences. In still other embodiments, thetarget region of the mRNA sequence is located in a 5′ untranslatedregion (UTR) or a 3′ UTR of the mRNA of a mutant protein.

In another embodiment, the invention provides an expression constructcomprising an isolated nucleic acid that encodes a nucleic acid moleculewith a first sequence of 16-25 nucleotides homologous to an allelicpolymorphism within, for example, the gene encoding a mutant huntingtinprotein. The expression construct can be for example, a viral vector,retroviral vector, expression cassette or plasmid. The expressionconstruct can also have an RNA polymerase II promoter sequence or RNAPolymerase II promoter sequence, such as, U6 snRNA promoter of H1promoter.

In yet other embodiments, the present invention provides host cellse.g.,) mammalian cells) comprising nucleic acid molecules and expressionconstructs of the present invention.

In still other embodiments, the present invention provides therapeuticcompositions comprising the nucleic acid molecules of the invention anda pharmaceutically acceptable carrier.

In some embodiments, the present invention utilizes RNA silencingtechnology (e.g. RNAi) against single nucleotide polymorphisms (SNPs)located within the htt gene encoding the mutant Huntington protein. RNAsilencing destroys the corresponding mutant mRNA with single nucleotidespecificity and selectivity. RNA silencing agents of the presentinvention are targeted to polymorphic regions of the mutant htt gene,resulting in cleavage or translational repression of mutant htt mRNA.These RNA silencing agents, through a series of protein-nucleotideinteractions, function to cleave or translationally repress the mutanthtt mRNAs.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. Additional exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine,²N-methylguanosine and ^(2,2)N,N-dimethylguanosine (also referred to as“rare” nucleosides). The term “nucleotide” refers to a nucleoside havingone or more phosphate groups joined in ester linkages to the sugarmoiety. Exemplary nucleotides include nucleoside monophosphates,diphosphates and triphosphates. The terms “polynucleotide” and “nucleicacid molecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30,or more ribonucleotides). The term “DNA” or “DNA molecule” ordeoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.Preferably, a siRNA comprises between about 15-30 nucleotides ornucleotide analogs, more preferably between about 16-25 nucleotides (ornucleotide analogs), even more preferably between about 18-23nucleotides (or nucleotide analogs), and even more preferably betweenabout 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22nucleotides or nucleotide analogs). The term “short” siRNA refers to asiRNA comprising ˜21 nucleotides (or nucleotide analogs), for example,19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNAcomprising ˜24-25 nucleotides, for example, 23, 24, 25 or 26nucleotides. Short siRNAs may, in some instances, include fewer than 19nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shortersiRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, insome instances, include more than 26 nucleotides, provided that thelonger siRNA retains the ability to mediate RNAi absent furtherprocessing, e.g., enzymatic processing, to a short siRNA.

The term “nucleotide analog” or “altered nucleotide” or “modifiednucleotide” refers to a non-standard nucleotide, including non-naturallyoccurring ribonucleotides or deoxyribonucleotides. Preferred nucleotideanalogs are modified at any position so as to alter certain chemicalproperties of the nucleotide yet retain the ability of the nucleotideanalog to perform its intended function. Examples of positions of thenucleotide which may be derivatized include the 5 position, e.g.,5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine,5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyluridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromoguanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotideanalogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- andN-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwiseknown in the art) nucleotides; and other heterocyclically modifiednucleotide analogs such as those described in Herdewijn, AntisenseNucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portionof the nucleotides. For example the 2′ OH-group may be replaced by agroup selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR,or OR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl,alkynyl, aryl, etc. Other possible modifications include those describedin U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., bysubstituting one or more of the oxygens of the phosphate group withsulfur (e.g., phosphorothioates), or by making other substitutions whichallow the nucleotide to perform its intended function such as describedin, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr.10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct.11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of theabove-referenced modifications (e.g., phosphate group modifications)preferably decrease the rate of hydrolysis of, for example,polynucleotides comprising said analogs in vivo or in vitro.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate,and/or phosphorothioate linkages. Preferred RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

An RNAi agent, e.g., an RNA silencing agent, having a strand which is“sequence sufficiently complementary to a target mRNA sequence to directtarget-specific RNA interference (RNAi)” means that the strand has asequence sufficient to trigger the destruction of the target mRNA by theRNAi machinery or process.

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or“isolated siRNA precursor”) refers to RNA molecules which aresubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, the term “RNA silencing” refers to a group ofsequence-specific regulatory mechanisms (e.g. RNA interference (RNAi),transcriptional gene silencing (TGS), post-transcriptional genesilencing (PTGS), quelling, co-suppression, and translationalrepression) mediated by RNA molecules which result in the inhibition or“silencing” of the expression of a corresponding protein-coding gene.RNA silencing has been observed in many types of organisms, includingplants, animals, and fungi.

The term “discriminatory RNA silencing” refers to the ability of an RNAmolecule to substantially inhibit the expression of a “first” or“target” polynucleotide sequence while not substantially inhibiting theexpression of a “second” or “non-target” polynucleotide sequence”, e.g.,when both polynucleotide sequences are present in the same cell. Incertain embodiments, the target polynucleotide sequence corresponds to atarget gene, while the non-target polynucleotide sequence corresponds toa non-target gene. In other embodiments, the target polynucleotidesequence corresponds to a target allele, while the non-targetpolynucleotide sequence corresponds to a non-target allele. In certainembodiments, the target polynucleotide sequence is the DNA sequenceencoding the regulatory region (e.g. promoter or enhancer elements) of atarget gene. In other embodiments, the target polynucleotide sequence isa target mRNA encoded by a target gene.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

As used herein, the term “transgene” refers to any nucleic acidmolecule, which is inserted by artifice into a cell, and becomes part ofthe genome of the organism that develops from the cell. Such a transgenemay include a gene that is partly or entirely heterologous (i.e.,foreign) to the transgenic organism, or may represent a gene homologousto an endogenous gene of the organism. The term “transgene” also means anucleic acid molecule that includes one or more selected nucleic acidsequences, e.g., DNAs, that encode one or more engineered RNAprecursors, to be expressed in a transgenic organism, e.g., animal,which is partly or entirely heterologous, i.e., foreign, to thetransgenic animal, or homologous to an endogenous gene of the transgenicanimal, but which is designed to be inserted into the animal's genome ata location which differs from that of the natural gene. A transgeneincludes one or more promoters and any other DNA, such as introns,necessary for expression of the selected nucleic acid sequence, alloperably linked to the selected sequence, and may include an enhancersequence.

A gene “involved” in a disease or disorder includes a gene, the normalor aberrant expression or function of which effects or causes thedisease or disorder or at least one symptom of said disease or disorder

The term “gain-of-function mutation” as used herein, refers to anymutation in a gene in which the protein encoded by said gene (i.e., themutant protein) acquires a function not normally associated with theprotein (i.e., the wild type protein) causes or contributes to a diseaseor disorder. The gain-of-function mutation can be a deletion, addition,or substitution of a nucleotide or nucleotides in the gene which givesrise to the change in the function of the encoded protein. In oneembodiment, the gain-of-function mutation changes the function of themutant protein or causes interactions with other proteins. In anotherembodiment, the gain-of-function mutation causes a decrease in orremoval of normal wild-type protein, for example, by interaction of thealtered, mutant protein with said normal, wild-type protein.

As used herein, the term “target gene” is a gene whose expression is tobe substantially inhibited or “silenced.” This silencing can be achievedby RNA silencing, e.g. by cleaving the mRNA of the target gene ortranslational repression of the target gene. The term “non-target gene”is a gene whose expression is not to be substantially silenced. In oneembodiment, the polynucleotide sequences of the target and non-targetgene (e.g. mRNA encoded by the target and non-target genes) can differby one or more nucleotides. In another embodiment, the target andnon-target genes can differ by one or more polymorphisms (e.g., SingleNucleotide Polymorphisms or SNPs). In another embodiment, the target andnon-target genes can share less than 100% sequence identity. In anotherembodiment, the non-target gene may be a homolog (e.g. an ortholog orparalog) of the target gene.

A “target allele” is an allele (e.g., a SNP allele) whose expression isto be selectively inhibited or “silenced.” This silencing can beachieved by RNA silencing, e.g. by cleaving the mRNA of the target geneor target allele by a siRNA. The term “non-target allele” is a allelewhose expression is not to be substantially silenced. In certainembodiments, the target and non-target alleles can correspond to thesame target gene. In other embodiments, the target allele correspondsto, or is associated with, a target gene, and the non-target allelecorresponds to, or is associated with, a non-target gene. In oneembodiment, the polynucleotide sequences of the target and non-targetalleles can differ by one or more nucleotides. In another embodiment,the target and non-target alleles can differ by one or more allelicpolymorphisms (e.g., one or more SNPs). In another embodiment, thetarget and non-target alleles can share less than 100% sequenceidentity.

The term “polymorphism” as used herein, refers to a variation (e.g., oneor more deletions, insertions, or substitutions) in a gene sequence thatis identified or detected when the same gene sequence from differentsources or subjects (but from the same organism) are compared. Forexample, a polymorphism can be identified when the same gene sequencefrom different subjects are compared. Identification of suchpolymorphisms is routine in the art, the methodologies being similar tothose used to detect, for example, breast cancer point mutations.Identification can be made, for example, from DNA extracted from asubject's lymphocytes, followed by amplification of polymorphic regionsusing specific primers to said polymorphic region. Alternatively, thepolymorphism can be identified when two alleles of the same gene arecompared. In particular embodiments, the polymorphism is a singlenucleotide polymorphism (SNP).

A variation in sequence between two alleles of the same gene within anorganism is referred to herein as an “allelic polymorphism”. In certainembodiments, the allelic polymorphism corresponds to a SNP allele. Forexample, the allelic polymorphism may comprise a single nucleotidevariation between the two alleles of a SNP. The polymorphism can be at anucleotide within a coding region but, due to the degeneracy of thegenetic code, no change in amino acid sequence is encoded.Alternatively, polymorphic sequences can encode a different amino acidat a particular position, but the change in the amino acid does notaffect protein function. Polymorphic regions can also be found innon-encoding regions of the gene. In preferred embodiments, thepolymorphism is found in a coding region of the gene or in anuntranslated region (e.g., a 5′ UTR or 3′ UTR) of the gene.

As used herein, the term “allelic frequency” is a measure (e.g.,proportion or percentage) of the relative frequency of an allele (e.g.,a SNP allele) at a single locus in a population of individuals. Forexample, where a population of individuals carry n loci of a particularchromosomal locus (and the gene occupying the locus) in each of theirsomatic cells, then the allelic frequency of an allele is the fractionor percentage of loci that the allele occupies within the population. Inparticular embodiments, the allelic frequency of an allele (e.g. a SNPallele) is at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40% ormore) in a sample population.

As used herein, the term “sample population” refers to a population ofindividuals comprising a statistically significant number ofindividuals. For example, the sample population may comprise 50, 75,100, 200, 500, 1000 or more individuals. In particular embodiments, thesample population may comprise individuals which share at least oncommon disease phenotype (e.g., a gain-of-function disorder) or mutation(e.g., a gain-of-function mutation).

As used herein, the term “heterozygosity” refers to the fraction ofindividuals within a population that are heterozygous (e.g., contain twoor more different alleles) at a particular locus (e.g., at a SNP).Heterozygosity may be calculated for a sample population using methodsthat are well known to those skilled in the art.

The term “polyglutamine domain,” as used herein, refers to a segment ordomain of a protein that consist of a consecutive glutamine residueslinked to peptide bonds. In one embodiment the consecutive regionincludes at least 5 glutamine residues.

The term “expanded polyglutamine domain” or “expanded polyglutaminesegment”, as used herein, refers to a segment or domain of a proteinthat includes at least 35 consecutive glutamine residues linked bypeptide bonds. Such expanded segments are found in subjects afflictedwith a polyglutamine disorder, as described herein, whether or not thesubject has shown to manifest symptoms.

The term “trinucleotide repeat” or “trinucleotide repeat region” as usedherein, refers to a segment of a nucleic acid sequence e.g.,) thatconsists of consecutive repeats of a particular trinucleotide sequence.In one embodiment, the trinucleotide repeat includes at least 5consecutive trinucleotide sequences. Exemplary trinucleotide sequencesinclude, but are not limited to, CAG, CGG, GCC, GAA, CTG, and/or CGG.

The term “trinucleotide repeat diseases” as used herein, refers to anydisease or disorder characterized by an expanded trinucleotide repeatregion located within a gene, the expanded trinucleotide repeat regionbeing causative of the disease or disorder. Examples of trinucleotiderepeat diseases include, but are not limited to spino-cerebellar ataxiatype 12 spino-cerebellar ataxia type 8, fragile X syndrome, fragile XEMental Retardation, Friedreich's ataxia and myotonic dystrophy.Preferred trinucleotide repeat diseases for treatment according to thepresent invention are those characterized or caused by an expandedtrinucleotide repeat region at the 5′ end of the coding region of agene, the gene encoding a mutant protein which causes or is causative ofthe disease or disorder. Certain trinucleotide diseases, for example,fragile X syndrome, where the mutation is not associated with a codingregion may not be suitable for treatment according to the methodologiesof the present invention, as there is no suitable mRNA to be targeted byRNAi. By contrast, disease such as Friedreich's ataxia may be suitablefor treatment according to the methodologies of the invention because,although the causative mutation is not within a coding region (i.e.,lies within an intron), the mutation may be within, for example, an mRNAprecursor (e.g., a pre-spliced mRNA precursor).

The term “polyglutamine disorder” as used herein, refers to any diseaseor disorder characterized by an expanded of a (CAG)_(n) repeats at the5′ end of the coding region (thus encoding an expanded polyglutamineregion in the encoded protein). In one embodiment, polyglutaminedisorders are characterized by a progressive degeneration of nervecells. Examples of polyglutamine disorders include but are not limitedto: Huntington's disease, spino-cerebellar ataxia type 1,spino-cerebellar ataxia type 2, spino-cerebellar ataxia type 3 (alsoknow as Machado-Joseph disease), and spino-cerebellar ataxia type 6,spino-cerebellar ataxia type 7 and dentatoiubral-pallidoluysian atrophy.

The phrase “examining the function of a gene in a cell or organism”refers to examining or studying the expression, activity, function orphenotype arising therefrom.

As used herein, the term “RNA silencing agent” refers to an RNA which iscapable of inhibiting or “silencing” the expression of a target gene. Incertain embodiments, the RNA silencing agent is capable of preventingcomplete processing (e.g, the full translation and/or expression) of amRNA molecule through a post-transcriptional silencing mechanism. RNAsilencing agents include small (<50 b.p.), noncoding RNA molecules, forexample RNA duplexes comprising paired strands, as well as precursorRNAs from which such small non-coding RNAs can be generated. ExemplaryRNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, anddual-function oligonucleotides as well as precursors thereof. In oneembodiment, the RNA silencing agent is capable of inducing RNAinterference. In another embodiment, the RNA silencing agent is capableof mediating translational repression.

As used herein, the term “rare nucleotide” refers to a naturallyoccurring nucleotide that occurs infrequently, including naturallyoccurring deoxyribonucleotides or ribonucleotides that occurinfrequently, e.g., a naturally occurring ribonucleotide that is notguanosine, adenosine, cytosine, or uridine. Examples of rare nucleotidesinclude, but are not limited to, inosine, 1-methyl inosine,pseudouridine, 5,6-dihydrouridine, ribothymidine, ²N-methylguanosine and^(2,2)N,N-dimethylguanosine.

The term “engineered,” as in an engineered RNA precursor, or anengineered nucleic acid molecule, indicates that the precursor ormolecule is not found in nature, in that all or a portion of the nucleicacid sequence of the precursor or molecule is created or selected byman. Once created or selected, the sequence can be replicated,translated, transcribed, or otherwise processed by mechanisms within acell. Thus, an RNA precursor produced within a cell from a transgenethat includes an engineered nucleic acid molecule is an engineered RNAprecursor.

As used herein, the term “microRNA” (“miRNA”), also referred to in theart as “small temporal RNAs” (“stRNAs”), refers to a small (10-50nucleotide) RNA which are genetically encoded (e.g. by viral, mammalian,or plant genomes) and are capable of directing or mediating RNAsilencing. An “miRNA disorder” shall refer to a disease or disordercharacterized by an aberrant expression or activity of an miRNA.

As used herein, the term “dual functional oligonucleotide” refers to aRNA silencing agent having the formula T-L-μ, wherein T is an mRNAtargeting moiety, L is a linking moiety, and μ is a miRNA recruitingmoiety. As used herein, the terms “mRNA targeting moiety”, “targetingmoiety”, “mRNA targeting portion” or “targeting portion” refer to adomain, portion or region of the dual functional oligonucleotide havingsufficient size and sufficient complementarity to a portion or region ofan mRNA chosen or targeted for silencing (i.e., the moiety has asequence sufficient to capture the target mRNA). As used herein, theterm “linking moiety” or “linking portion” refers to a domain, portionor region of the RNA-silencing agent which covalently joins or links themRNA.

As used herein, the term “antisense strand” of an RNA silencing agent,e.g. an siRNA or RNA silencing agent, refers to a strand that issubstantially complementary to a section of about 10-50 nucleotides,e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of thegene targeted for silencing. The antisense strand or first strand hassequence sufficiently complementary to the desired target mRNA sequenceto direct target-specific silencing, e.g., complementarity sufficient totrigger the destruction of the desired target mRNA by the RNAi machineryor process (RNAi interference) or complementarity sufficient to triggertranslational repression of the desired target mRNA.

The term “sense strand” or “second strand” of an RNA silencing agent,e.g. an siRNA or RNA silencing agent, refers to a strand that iscomplementary to the antisense strand or first strand. Antisense andsense strands can also be referred to as first or second strands, thefirst or second strand having complementarity to the target sequence andthe respective second or first strand having complementarity to saidfirst or second strand. miRNA duplex intermediates or siRNA-likeduplexes include a miRNA strand having sufficient complementarity to asection of about 10-50 nucleotides of the mRNA of the gene targeted forsilencing and a miRNA* strand having sufficient complementarity to forma duplex with the miRNA strand.

As used herein, the term “guide strand” refers to a strand of an RNAsilencing agent, e.g., an antisense strand of an siRNA duplex or siRNAsequence, that enters into the RISC complex and directs cleavage of thetarget mRNA.

As used herein, the term “asymmetry”, as in the asymmetry of the duplexregion of an RNA silencing agent (e.g. the stem of an shRNA), refers toan inequality of bond strength or base pairing strength between thetermini of the RNA silencing agent (e.g., between terminal nucleotideson a first strand or stem portion and terminal nucleotides on anopposing second strand or stem portion), such that the 5′ end of onestrand of the duplex is more frequently in a transient unpaired, e.g,single-stranded, state than the 5′ end of the complementary strand. Thisstructural difference determines that one strand of the duplex ispreferentially incorporated into a RISC complex. The strand whose 5′ endis less tightly paired to the complementary strand will preferentiallybe incorporated into RISC and mediate RNAi.

As used herein, the term “bond strength” or “base pair strength” refersto the strength of the interaction between pairs of nucleotides (ornucleotide analogs) on opposing strands of an oligonucleotide duplex(e.g., an siRNA duplex), due primarily to H-bonding, Van der Waalsinteractions, and the like between said nucleotides (or nucleotideanalogs).

As used herein, the “5′ end”, as in the 5′ end of an antisense strand,refers to the 5′ terminal nucleotides, e.g., between one and about 5nucleotides at the 5′ terminus of the antisense strand. As used herein,the “3′ end”, as in the 3′ end of a sense strand, refers to the region,e.g., a region of between one and about 5 nucleotides, that iscomplementary to the nucleotides of the 5′ end of the complementaryantisense strand.

As used herein the term “destabilizing nucleotide” refers to a firstnucleotide or nucleotide analog capable of forming a base pair withsecond nucleotide or nucleotide analog such that the base pair is oflower bond strength than a conventional base pair (ie. Watson-Crick basepair). In certain embodiments, the destabilizing nucleotide is capableof forming a mismatch base pair with the second nucleotide. In otherembodiments, the destabilizing nucleotide is capable of forming a wobblebase pair with the second nucleotide. In yet other embodiments, thedestabilizing nucleotide is capable of forming an ambiguous base pairwith the second nucleotide.

As used herein, the term “base pair” refers to the interaction betweenpairs of nucleotides (or nucleotide analogs) on opposing strands of anoligonucleotide duplex (e.g., a duplex formed by a strand of a RNAsilencing agent and a target mRNA sequence), due primarily to H-bonding,Van der Waals interactions, and the like between said nucleotides (ornucleotide analogs). As used herein, the term “bond strength” or “basepair strength” refers to the strength of the base pair.

As used herein, the term “mismatched base pair” refers to a base pairconsisting of noncomplementary or non-Watson-Crick base pairs, forexample, not normal complementary G:C, A:T or A:U base pairs. As usedherein the term “ambiguous base pair” (also known as anon-discriminatory base pair) refers to a base pair formed by auniversal nucleotide.

As used herein, term “universal nucleotide” (also known as a “neutralnucleotide”) include those nucleotides (e.g. certain destabilizingnucleotides) having a base (a “universal base” or “neutral base”) thatdoes not significantly discriminate between bases on a complementarypolynucleotide when forming a base pair. Universal nucleotides arepredominantly hydrophobic molecules that can pack efficiently intoantiparallel duplex nucleic acids (e.g. double-stranded DNA or RNA) dueto stacking interactions. The base portion of universal nucleotidestypically comprise a nitrogen-containing aromatic heterocyclic moiety.

As used herein, the terms “sufficient complementarity” or “sufficientdegree of complementarity” mean that the RNA silencing agent has asequence (e.g. in the antisense strand, mRNA targeting moiety or miRNArecruiting moiety) which is sufficient to bind the desired target RNA,respectively, and to trigger the RNA silencing of the target mRNA.

As used herein, the term “translational repression” refers to aselective inhibition of mRNA translation. Natural translationalrepression proceeds via miRNAs cleaved from shRNA precursors. Both RNAiand translational repression are mediated by RISC. Both RNAi andtranslational repression occur naturally or can be initiated by the handof man, for example, to silence the expression of target genes.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing an RNA silencing agent of the invention into a cellor organism. In another embodiment, a “suitable control” or “appropriatecontrol” is a value, level, feature, characteristic, property, etc.determined in a cell or organism, e.g., a control or normal cell ororganism, exhibiting, for example, normal traits. In yet anotherembodiment, a “suitable control” or “appropriate control” is apredefined value, level, feature, characteristic, property, etc.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Polyglutamine Disorders

Polyglutamine disorders are a class of disease or disorderscharacterized by a common genetic mutation. In particular, the diseaseor disorders are characterized by an expanded repeat of thetrinucleotide CAG which gives rise, in the encoded protein, to anexpanded stretch of glutamine residues. Polyglutamine disorders aresimilar in that the diseases are characterized by a progressivedegeneration of nerve cells. Despite their similarities, polyglutaminedisorders occur on different chromosomes and thus occur on entirelydifferent segments of DNA. Examples of polyglutamine disorders includeHuntington's disease, Dentatorubropallidoluysian Atrophy, SpinobulbarMuscular atrophy, Spinocerebellar Ataxia Type 1, Spinocerebellar AtaxiaType 2, Spinocerebellar Ataxia Type 3, Spinocerebellar Ataxia Type 6 andSpinocerebellar Ataxia Type 7 (Table 3).

TABLE 1 Polyglutamine disorders CAG repeat size Disease Gene LocusProtein Normal Disease Spinobulbar AR Xq13-21 Androgen 9-36 38-62muscular receptor (AR) atrophy (Kennedy disease) Huntington's HD 4p16.3Huntingtin 6-35  36-121 disease Dentatorubral- DRPLA 12p13.31 Atrophin-16-35 49-88 pallidoluysian atrophy (Haw- River syndrome) SpinocerebellarSCA1 6p23 Ataxin-1  6-44^(a) 39-82 ataxia type 1 Spinocerebellar SCA212q24.1 Ataxin-2 15-31  36-63 ataxia type 2 Spinocerebellar SCA3 (MJD1)14q32.1 Ataxin-3 12-40  55-84 ataxia type 3 (Machado- Joseph disease)Spinocerebellar SCA6 19p13 α_(1A)-voltage- 4-18 21-33 ataxia type 6dependent calcium channel subunit Spinocerebellar SCA7 13p12-13 Ataxin-74-35  37-306 ataxia type 7 ^(a)Alleles with 21 or more repeats areinterrupted by 1-3 CAT units; disease alleles contain pure CAG tracts.

Polyglutamine disorders of the invention are characterized by (e.g.,domains having between about 30 to 35 glutamine residues, between about35 to 40 glutamine residues, between about 40 to 45 glutamine residuesand having about 45 or more glutamine residues. The polyglutamine domaintypically contains consecutive glutamine residues (Q n>36).

II. Huntington Disease

In some embodiments, the RNA silencing agents of the invention aredesigned to target polymorphisms (e.g. single nucleotide polymorphisms)in the mutant human huntingtin protein (htt) for the treatment ofHuntington's disease.

Huntington's disease, inherited as an autosomal dominant disease, causesimpaired cognition and motor disease. Patients can live more than adecade with severe debilitation, before premature death from starvationor infection. The disease begins in the fourth or fifth decade for mostcases, but a subset of patients manifest disease in teenage years. Thegenetic mutation for Huntington's disease is a lengthened CAG repeat inthe huntingtin gene. CAG repeat varies in number from 8 to 35 in normalindividuals (Kremer et al., 1994). The genetic mutation e.g.,) anincrease in length of the CAG repeats from normal less than 36 in thehuntingtin gene to greater than 36 in the disease is associated with thesynthesis of a mutant huntingtin protein, which has greater than 36polyglutamates (Aronin et al., 1995). In general, individuals with 36 ormore CAG repeats will get Huntington's disease. Prototypic for as manyas twenty other diseases with a lengthened CAG as the underlyingmutation, Huntington's disease still has no effective therapy. A varietyof interventions—such as interruption of apoptotic pathways, addition ofreagents to boost mitochondrial efficiency, and blockade of NMDAreceptors—have shown promise in cell cultures and mouse model ofHuntington's disease. However, at best these approaches reveal a shortprolongation of cell or animal survival.

Huntington's disease complies with the central dogma of genetics: amutant gene serves as a template for production of a mutant mRNA; themutant mRNA then directs synthesis of a mutant protein (Aronin et al.,1995; DiFiglia et al., 1997). Mutant huntingtin (protein) probablyaccumulates in selective neurons in the striatum and cortex, disrupts asyet determined cellular activities, and causes neuronal dysfunction anddeath (Aronin et al., 1999; Laforet et al., 2001). Because a single copyof a mutant gene suffices to cause Huntington's disease, the mostparsimonious treatment would render the mutant gene ineffective.Theoretical approaches might include stopping gene transcription ofmutant huntingtin, destroying mutant mRNA, and blocking translation.Each has the same outcome—loss of mutant huntingtin.

III. Huntingtin Gene

The disease gene linked to Huntington's disease is termed Huntington or(htt). The huntingtin locus is large, spanning 180 kb and consisting of67 exons. The huntingtin gene is widely expressed and is required fornormal development. It is expressed as 2 alternatively polyadenylatedforms displaying different relative abundance in various fetal and adulttissues. The larger transcript is approximately 13.7 kb and is expressedpredominantly in adult and fetal brain whereas the smaller transcript ofapproximately 10.3 kb is more widely expressed. The two transcriptsdiffer with respect to their 3′ untranslated regions (Lin et al., 1993).Both messages are predicted to encode a 348 kilodalton proteincontaining 3144 amino acids. The genetic defect leading to Huntington'sdisease is believed to confer a new property on the mRNA or alter thefunction of the protein. The amino acid sequence of the human huntingtinprotein is set forth in FIG. 2 (SEQ ID NO:2).

A consensus nucleotide sequence of the human huntingtin gene (cDNA) isset forth in FIG. 1 (SEQ ID NO:1). The coding region consists ofnucleotides 316 to 9750 of SEQ ID NO:1. The two alternativepolyadenylation signals are found at nucleotides 10326 to 10331 andnucleotides 13644 to 13649, respectively. The corresponding twopolyadenylation sites are found at nucleotides 10348 and 13672,respectively. The first polyadenylation signal/site is that of the 10.3kb transcript. The second polyadenylation signal/site is that of the13.7 kb transcript, the predominant transcript in brain.

Five (5) polymorphisms in the human htt gene were identified asdescribed in Example I. An additional 38 polymorphisms in the huntingtingene sequence have been identified via SNP (single nucleotidepolymorphism) analysis (see Table 3). The polymorphisms set forth inTables 2 and 3 represent preferred sites to target viasingle-nucleotide-specific RNAi, as described herein.

TABLE 2 Polymorphic sites (P) in the htt gene of human cell lines. Cellline P1 (2886) P2 (4034) P3 (6912) P4 (7222) P5 (7246) GFP-Htt C G A T C(9kb construct) HeLa t a A g C HEK 293T t a G g t HepG2 t a G g t FP-4 ta g, A g t, C

TABLE 3 Polymorphic sites (P) in the human htt gene identified by SNPanalysis. consensus polymorphism db_xref complement 103 G A P6 dbSNP:396875 complement 432 T C P7 dbSNP: 473915 complement 474 C A P8 dbSNP:603765 1509 T C P9 dbSNP: 1065745 complement 1857 T C P10 dbSNP: 23013673565 G C, A P11, P12 dbSNP: 1065746 3594 T G P13 dbSNP: 1143646 3665 G CP14 dbSNP: 1065747 complement 4122 G A P15 dbSNP: 363099 complement 4985G A P16 dbSNP: 363129 complement 5480 T G P17 dbSNP: 363125 6658 T G P18dbSNP: 1143648 complement 6912 T C P19 dbSNP: 362336 complement 7753 G AP20 dbSNP: 3025816 complement 7849 G C P21 dbSNP: 3025814 complement8478 T C P22 dbSNP: 2276881 8574 T C P23 dbSNP: 2229985 complement 9154C A P24 dbSNP: 3025807 9498 T C P25 dbSNP: 2229987 complement 9699 G AP26 dbSNP: 362308 complement 9809 G A P27 dbSNP: 362307 complement 10064T C P28 dbSNP: 362306 complement 10112 G C P29 dbSNP: 362268 complement10124 G C P30 dbSNP: 362305 complement 10236 T G P31 dbSNP: 362304complement 10271 G A P32 dbSNP: 362303 complement 10879 G A P33 dbSNP:1557210 complement 10883 G A P34 dbSNP: 362302 complement 10971 C A P35dbSNP: 3025805 complement 11181 G A P36 dbSNP: 362267 complement 11400 CA P37 dbSNP: 362301 11756 . . . 11757 G — P38 dbSNP: 5855774 12658 G AP39 dbSNP: 2237008 complement 12911 T C P40 dbSNP: 362300 complement13040 G A P41 dbSNP: 2530595 13482 G A P42 dbSNP: 1803770 13563 G A P43dbSNP: 1803771

The present invention targets mutant huntingtin using RNA interference(Hutvagner et al., 2002). One strand of double-stranded RNA (siRNA)complements a polymorphic region within the mutant huntingtin mRNA.After introduction of siRNA into neurons, the siRNA partially unwinds,binds to polymorphic region within the huntingtin mRNA in asite-specific manner, and activates an mRNA nuclease. This nucleasecleaves the huntingtin mRNA, thereby halting translation of the mutanthuntingtin. Cells rid themselves of partially digested mRNA, thusprecluding translation, or cells digest partially translated proteins.Neurons survive on the wild-type huntingtin (from the normal allele);this approach prevents the ravages of mutant huntingtin by eliminatingits production.

Exemplary single nucleotide polymorphisms in the huntingtin genesequence can be found at positions 2886, 4034, 6912, 7222, and 7246 ofthe human htt gene. In certain embodiments, RNA silencing agents of theinvention are capable of targeting one of the SNP sites listed in FIG.7. Genomic sequence for each SNP site can be found in, for example, thepublically available “SNP Entrez” database maintained by the NCBI.Additional single nucleotide polymorphisms in the huntingtin genesequence are set forth in Table 3.

In some embodiments, preferred htt SNPs have an allelic frequency of atleast 30% in a sample population of patients. In some embodiments, atargeted htt SNP exhibits a frequency of heterozygosity of at least 25%within a sample patient population (e.g., at least 30, 40, 50, 60, 65,70, 75, or 80% heterozygosity).

In a particular embodiment, the SNP allele is present at genomic siteRS363125. In another embodiment, the SNP allele is present at genomicsite RS362331. In another embodiment, the SNP allele is present atposition 171, e.g., an A171C polymorphism, in the huntingtin geneaccording to the sequence numbering in GenBank Accession No.NM_(—)002111 (Aug. 8, 2005).

IV. siRNA Design

In some embodiments, siRNAs are designed as follows. First, a portion ofthe target gene (e.g., the htt gene) is selected that includes thepolymorphism. Exemplary polymorphisms are selected from the 5′untranslated region of a target gene. Cleavage of mRNA at these sitesshould eliminate translation of corresponding mutant protein.Polymorphisms from other regions of the mutant gene are also suitablefor targeting. A sense strand is designed based on the sequence of theselected portion. Preferably the portion (and corresponding sensestrand) includes about 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23,24 or 25 nucleotides. More preferably, the portion (and correspondingsense strand) includes 21, 22 or 23 nucleotides. The skilled artisanwill appreciate, however, that siRNAs having a length of less than 19nucleotides or greater than 25 nucleotides can also function to mediateRNAi. Accordingly, siRNAs of such length are also within the scope ofthe instant invention provided that they retain the ability to mediateRNAi. Longer RNAi agents have been demonstrated to ellicit an interferonor PKR response in certain mammalian cells which may be undesirable.Preferably the RNAi agents of the invention do not ellicit a PKRresponse (i.e., are of a sufficiently short length). However, longerRNAi agents may be useful, for example, in cell types incapable ofgenerating a PRK response or in situations where the PKR response hasbeen downregulated or dampened by alternative means.

The sense strand sequence is designed such that the polymorphism isessentially in the middle of the strand. For example, if a 21-nucleotidesiRNA is chosen, the polymorphism is at, for example, nucleotide 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or 16 (i.e., 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16 nucleotides from the 5′ end of the sense strand. For a22-nucleotide siRNA, the polymorphism is at, for example, nucleotide 7,8, 9, 10, 11, 12, 13, 14, 15 or 16. For a 23-nucleotide siRNA, thepolymorphism is at, for example, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.For a 24-nucleotide siRNA, the polymorphism is at, for example, 9, 10,11, 12, 13, 14 or 16. For a 25-nucleotide siRNA, the polymorphism is at,for example, 9, 10, 11, 12, 13, 14, 15, 16 or 17. Moving thepolymorphism to an off-center position may, in some instances, reduceefficiency of cleavage by the siRNA. Such compositions, i.e., lessefficient compositions, may be desirable for use if off-silencing of thewild-type mRNA is detected.

The antisense strand is routinely the same length as the sense strandand include complementary nucleotides. In one embodiment, the strandsare fully complementary, i.e., the strands are blunt-ended when alignedor annealed. In another embodiment, the strands comprise align or annealsuch that 1-, 2- or 3-nucleotide overhangs are generated, i.e., the 3′end of the sense strand extends 1, 2 or 3 nucleotides further than the5′ end of the antisense strand and/or the 3′ end of the antisense strandextends 1, 2 or 3 nucleotides further than the 5′ end of the sensestrand. Overhangs can comprise (or consist of) nucleotides correspondingto the target gene sequence (or complement thereof). Alternatively,overhangs can comprise (or consist of) deoxyribonucleotides, for exampledTs, or nucleotide analogs, or other suitable non-nucleotide material.

To facilitate entry of the antisense strand into RISC (and thus increaseor improve the efficiency of target cleavage and silencing), the basepair strength between the 5′ end of the sense strand and 3′ end of theantisense strand can be altered, e.g., lessened or reduced, as describedin detail in U.S. Provisional patent application Nos. 60/475,386entitled “Methods and Compositions for Controlling Efficacy of RNASilencing” (filed Jun. 2, 2003) and 60/475,331 entitled “Methods andCompositions for Enhancing the Efficacy and Specificity of RNAi” (filedJun. 2, 2003), the contents of which are incorporated in their entiretyby this reference. In one embodiment of these aspects of the invention,the base-pair strength is less due to fewer G:C base pairs between the5′ end of the first or antisense strand and the 3′ end of the second orsense strand than between the 3′ end of the first or antisense strandand the 5′ end of the second or sense strand. In another embodiment, thebase pair strength is less due to at least one mismatched base pairbetween the 5′ end of the first or antisense strand and the 3′ end ofthe second or sense strand. Preferably, the mismatched base pair isselected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C andU:U. In another embodiment, the base pair strength is less due to atleast one wobble base pair, e.g., G:U, between the 5′ end of the firstor antisense strand and the 3′ end of the second or sense strand. Inanother embodiment, the base pair strength is less due to at least onebase pair comprising a rare nucleotide, e.g., inosine (I). Preferably,the base pair is selected from the group consisting of an I:A, I:U andI:C. In yet another embodiment, the base pair strength is less due to atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

The design of siRNAs suitable for targeting the htt polymorphisms setforth in Table 2 is described in detail below

P1 DNA TGTGCTGAC

CTGAGGAACAG                                (SEQ ID NO: 5) senseUGUGCUGAC

CUGAGGAACAG (SEQ ID NO: 6) anti- ACACGACUGAGACUCCUUGUC (blunt-ends,sense 21-mer) (SEQ ID NO: 7) (2-nt overhangs) see FIG. 5 P2 DNACATACCTCA

ACTGCATGATG (SEQ ID NO: 8) sense CAUACCUCA

ACUGCAUGAUG (SEQ ID NO: 9) anti- GUAUGGAGUUUGACGUACUAC (blunt ends,sense 21-mer) (SEQ ID NO: 10) P3 DNA GCCTGCAGA

CCGGCGGCCTA (SEQ ID NO: 11) sense GCCUGCAGA

CCGGCGGCCUA (SEQ ID NO: 12) anti- CGGACGUCUCGGCCGCCGGAU (blunt ends,sense 21-mer) (SEQ ID NO: 13) P4 DNA ACAGAGTTT

TGACCCACGCC (SEQ ID NO: 14) sense ACAGAGUUU

UGACCCACGCC (SEQ ID NO: 15) anti- UGUCUCAAACACUGGGUGCGG (blunt ends,sense 21-mer) (SEQ ID NO: 16) P5 DNA TCCCTCATC

ACTGTGTGCAC (SEQ ID NO: 17) sense UCCCUCAUC

ACUGUGUGCAC (SEQ ID NO: 18) anti- AGGGAGUAGAUGACACACGUG (blunt ends,sense 21 mer) (SEQ ID NO: 19)

siRNAs can be designed according to the above exemplary teachings forany other polymorphisms found in the htt gene. Moreover, the technologyis applicable to targeting any other disease gene having associatedpolymorphisms, i.e., non-disease causing polymorphisms.

To validate the effectiveness by which siRNAs destroy mutant mRNAs(e.g., mutant huntingtin mRNA), the siRNA is incubated with mutant cDNA(e.g., mutant huntingtin cDNA) in a Drosophila-based in vitro mRNAexpression system. Radiolabeled with ³²P, newly synthesized mutant mRNAs(e.g., mutant huntingtin mRNA) are detected autoradiographically on anagarose gel. The presence of cleaved mutant mRNA indicates mRNA nucleaseactivity. Suitable controls include omission of siRNA and use ofwild-type huntingtin cDNA. Alternatively, control siRNAs are selectedhaving the same nucleotide composition as the selected siRNA, butwithout significant sequence complementarity to the appropriate targetgene. Such negative controls can be designed by randomly scrambling thenucleotide sequence of the selected siRNA; a homology search can beperformed to ensure that the negative control lacks homology to anyother gene in the appropriate genome. In addition, negative controlsiRNAs can be designed by introducing one or more base mismatches intothe sequence.

Sites of siRNA-mRNA complementation are selected which result in optimalmRNA specificity and maximal mRNA cleavage.

While the instant invention primarily features targeting polymorphicregions in the target mutant gene (e.g., in mutant htt) distinct fromthe expanded CAG region mutation, the skilled artisan will appreciatethat targeting the mutant region may have applicability as a therapeuticstrategy in certain situations. Targeting the mutant region can beaccomplished using siRNA that complements CAG in series. The siRNA^(cag)would bind to mRNAs with CAG complementation, but might be expected tohave greater opportunity to bind to an extended CAG series. MultiplesiRNA^(cag) would bind to the mutant huntingtin mRNA (as opposed tofewer for the wild type huntingtin mRNA); thus, the mutant huntingtinmRNA is more likely to be cleaved. Successful mRNA inactivation usingthis approach would also eliminate normal or wild-type huntingtin mRNA.Also inactivated, at least to some extent, could be other normal genes(approximately 70) which also have CAG repeats, where their mRNAs couldinteract with the siRNA. This approach would thus rely on an attritionstrategy—more of the mutant huntingtin mRNA would be destroyed than wildtype huntingtin mRNA or the other approximately 69 mRNAs that code forpolyglutamines.

V. RNAi Agents

The present invention includes siRNA molecules designed, for example, asdescribed above. The siRNA molecules of the invention can be chemicallysynthesized, or can be transcribed in vitro from a DNA template, or invivo from e.g., shRNA, or, by using recombinant human DICER enzyme, tocleave in vitro transcribed dsRNA templates into pools of 20- , 21- or23- bp duplex RNA mediating RNAi. The siRNA molecules can be designedusing any method known in the art.

In one aspect, instead of the RNAi agent being an interferingribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAiagent can encode an interfering ribonucleic acid, e.g., an shRNA, asdescribed above. In other words, the RNAi agent can be a transcriptionaltemplate of the interfering ribonucleic acid. Thus, RNAi agents of thepresent invention can also include small hairpin RNAs (shRNAs), andexpression constructs engineered to express shRNAs. Transcription ofshRNAs is initiated at a polymerase III (pol III) promoter, and isthought to be terminated at position 2 of a 4-5-thymine transcriptiontermination site. Upon expression, shRNAs are thought to fold into astem-loop structure with 3′ UU-overhangs; subsequently, the ends ofthese shRNAs are processed, converting the shRNAs into siRNA-likemolecules of about 21-23 nucleotides (Brummelkamp et al., 2002; Lee etal., 2002. supra; Miyagishi et al., 2002; Paddison et al., 2002, supra;Paul et al., 2002, supra; Sui et al., 2002 supra; Yu et al., 2002,supra. More information about shRNA design and use can be found on theinternet at the following addresses:katandin.cshl.org:9331/RNAi/docs/BseRI-BamHI_Strategy.pdf andkatandin.cshl.org:9331/RNAi/docs/Web_version_of_PCR_strategy1.pdf.

Expression constructs of the present invention include any constructsuitable for use in the appropriate expression system and include, butare not limited to, retroviral vectors, linear expression cassettes,plasmids and viral or virally-derived vectors, as known in the art. Suchexpression constructs can include one or more inducible promoters, RNAPol III promoter systems such as U6 snRNA promoters or H1 RNA polymeraseIII promoters, or other promoters known in the art. The constructs caninclude one or both strands of the siRNA. Expression constructsexpressing both strands can also include loop structures linking bothstrands, or each strand can be separately transcribed from separatepromoters within the same construct. Each strand can also be transcribedfrom a separate expression construct. (Tuschl, T., 2002, supra).

Synthetic siRNAs can be delivered into cells by methods known in theart, including cationic liposome transfection and electroporation.However, these exogenous siRNA generally show short term persistence ofthe silencing effect (4-5 days in cultured cells), which may bebeneficial in only certain embodiments. To obtain longer termsuppression of the target genes (i.e., mutant genes) and to facilitatedelivery under certain circumstances, one or more siRNA can be expressedwithin cells from recombinant DNA constructs. Such methods forexpressing siRNA duplexes within cells from recombinant DNA constructsto allow longer-term target gene suppression in cells are known in theart, including mammalian Pol III promoter systems (e.g., H1 or U6/snRNApromoter systems (Tuschl, T., 2002, supra) capable of expressingfunctional double-stranded siRNAs; (Bagella et al., 1998; Lee et al.,2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yuet al., 2002), supra; Sui et al., 2002, supra). Transcriptionaltermination by RNA Pol III occurs at runs of four consecutive T residuesin the DNA template, providing a mechanism to end the siRNA transcriptat a specific sequence. The siRNA is complementary to the sequence ofthe target gene in 5′-3′ and 3′-5′ orientations, and the two strands ofthe siRNA can be expressed in the same construct or in separateconstructs. Hairpin siRNAs, driven by H1 or U6 snRNA promoter andexpressed in cells, can inhibit target gene expression (Bagella et al.,1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul etal., 2002, supra; Yu et al., 2002), supra; Sui et al., 2002, supra).Constructs containing siRNA sequence under the control of T7 promoteralso make functional siRNAs when cotransfected into the cells with avector expressing T7 RNA polymerase (Jacque et al., 2002, supra). Asingle construct may contain multiple sequences coding for siRNAs, suchas multiple regions of the gene encoding mutant htt, targeting the samegene or multiple genes, and can be driven, for example, by separatePolIII promoter sites.

Animal cells express a range of noncoding RNAs of approximately 22nucleotides termed micro RNA (miRNAs) which can regulate gene expressionat the post transcriptional or translational level during animaldevelopment. One common feature of miRNAs is that they are all excisedfrom an approximately 70 nucleotide precursor RNA stem-loop, probably byDicer, an RNase III-type enzyme, or a homolog thereof. By substitutingthe stem sequences of the miRNA precursor with sequence complementary tothe target mRNA, a vector construct that expresses the engineeredprecursor can be used to produce siRNAs to initiate RNAi againstspecific mRNA targets in mammalian cells (Zeng et al., 2002, supra).When expressed by DNA vectors containing polymerase III promoters,micro-RNA designed hairpins can silence gene expression (McManus et al.,2002, supra). MicroRNAs targeting polymorphisms may also be useful forblocking translation of mutant proteins, in the absence ofsiRNA-mediated gene-silencing. Such applications may be useful insituations, for example, where a designed siRNA caused off-targetsilencing of wild type protein.

Viral-mediated delivery mechanisms can also be used to induce specificsilencing of targeted genes through expression of siRNA, for example, bygenerating recombinant adenoviruses harboring siRNA under RNA Pol IIpromoter transcription control (Xia et al., 2002, supra). Infection ofHeLa cells by these recombinant adenoviruses allows for diminishedendogenous target gene expression. Injection of the recombinantadenovirus vectors into transgenic mice expressing the target genes ofthe siRNA results in in vivo reduction of target gene expression. Id. Inan animal model, whole-embryo electroporation can efficiently deliversynthetic siRNA into post-implantation mouse embryos (Calegari et al.,2002). In adult mice, efficient delivery of siRNA can be accomplished by“high-pressure” delivery technique, a rapid injection (within 5 seconds)of a large volume of siRNA containing solution into animal via the tailvein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis etal., 2002. Nanoparticles and liposomes can also be used to deliver siRNAinto animals.

The nucleic acid compositions of the invention include both unmodifiedsiRNAs and modified siRNAs as known in the art, such as crosslinkedsiRNA derivatives or derivatives having non nucleotide moieties linked,for example to their 3′ or 5′ ends. Modifying siRNA derivatives in thisway may improve cellular uptake or enhance cellular targeting activitiesof the resulting siRNA derivative as compared to the correspondingsiRNA, are useful for tracing the siRNA derivative in the cell, orimprove the stability of the siRNA derivative compared to thecorresponding siRNA.

Engineered RNA precursors, introduced into cells or whole organisms asdescribed herein, will lead to the production of a desired siRNAmolecule. Such an siRNA molecule will then associate with endogenousprotein components of the RNAi pathway to bind to and target a specificmRNA sequence for cleavage and destruction. In this fashion, the mRNA tobe targeted by the siRNA generated from the engineered RNA precursorwill be depleted from the cell or organism, leading to a decrease in theconcentration of the protein encoded by that mRNA in the cell ororganism. The RNA precursors are typically nucleic acid molecules thatindividually encode either one strand of a dsRNA or encode the entirenucleotide sequence of an RNA hairpin loop structure.

The nucleic acid compositions of the invention can be unconjugated orcan be conjugated to another moiety, such as a nanoparticle, to enhancea property of the compositions, e.g., a pharmacokinetic parameter suchas absorption, efficacy, bioavailability, and/or half-life. Theconjugation can be accomplished by methods known in the art, e.g., usingthe methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001)(describes nucleic acids loaded to polyalkylcyanoacrylate (PACA)nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998)(describes nucleic acids bound to nanoparticles); Schwab et al., Ann.Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked tointercalating agents, hydrophobic groups, polycations or PACAnanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995)(describes nucleic acids linked to nanoparticles).

The nucleic acid molecules of the present invention can also be labeledusing any method known in the art; for instance, the nucleic acidcompositions can be labeled with a fluorophore, e.g., Cy3, fluorescein,or rhodamine. The labeling can be carried out using a kit, e.g., theSILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can beradiolabeled, e.g., using ³H, ³²P, or other appropriate isotope.

Moreover, because RNAi is believed to progress via at least onesingle-stranded RNA intermediate, the skilled artisan will appreciatethat ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also bedesigned (e.g., for chemical synthesis) generated (e.g., enzymaticallygenerated) or expressed (e.g., from a vector or plasmid) as describedherein and utilized according to the claimed methodologies. Moreover, ininvertebrates, RNAi can be triggered effectively by long dsRNAs (e.g.,dsRNAs about 100-1000 nucleotides in length, preferably about 200-500,for example, about 250, 300, 350, 400 or 450 nucleotides in length)acting as effectors of RNAi. (Brondani et al., Proc Natl Acad Sci USA.2001 Dec. 4; 98(25):14428-33. Epub 2001 Nov. 27).

VI. Anti-Htt RNA Silencing Agents

The present invention features anti-huntingtin RNA silencing agents(e.g., siRNA and shRNAs), methods of making said RNA silencing agents,and methods (e.g., research and/or therapeutic methods) for using saidimproved RNA silencing agents (or portions thereof) for RNA silencing ofmutant huntingtin protein. The RNA silencing agents comprise anantisense strand (or portions thereof), wherein the antisense strand hassufficient complementary to a heterozygous single nucleotidepolymorphism to mediate an RNA-mediated silencing mechanism (e.g. RNAi).

a) Design of Anti-Htt siRNA Molecules

An siRNA molecule of the invention is a duplex consisting of a sensestrand and complementary antisense strand, the antisense strand havingsufficient complementary to a htt mRNA to mediate RNAi. Preferably, thesiRNA molecule has a length from about 10-50 or more nucleotides, i.e.,each strand comprises 10-50 nucleotides (or nucleotide analogs). Morepreferably, the siRNA molecule has a length from about 16-30, e.g., 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides ineach strand, wherein one of the strands is sufficiently complementary toa target region. Preferably, the strands are aligned such that there areat least 1, 2, or 3 bases at the end of the strands which do not align(i.e., for which no complementary bases occur in the opposing strand)such that an overhang of 1, 2 or 3 residues occurs at one or both endsof the duplex when strands are annealed. Preferably, the siRNA moleculehas a length from about 10-50 or more nucleotides, i.e., each strandcomprises 10-50 nucleotides (or nucleotide analogs). More preferably,the siRNA molecule has a length from about 16-30, e.g., 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in eachstrand, wherein one of the strands is substantially complementary to atarget region e.g., a gain-of-function gene target region, and the otherstrand is identical or substantially identical to the first strand.

Generally, siRNAs can be designed by using any method known in the art,for instance, by using the following protocol:

1. The siRNA should be specific for a heterozygous single-nucleotidepolymorphism (SNP) found in a mutant huntingtin (htt) allele, but not awild-type huntingtin allele. The first strand should be complementary tothis sequence, and the other strand is substantially complementary tothe first strand. In one embodiment, the SNP is outside the expanded CAGrepeat of the mutant huntingin (htt) allele. In another embodiment, theSNP is outside a coding region of the target gene. Exemplarypolymorphisms are selected from the 5′ untranslated region (5′-UTR) of atarget gene. Cleavage of mRNA at these sites should eliminatetranslation of corresponding mutant protein. Polymorphisms from otherregions of the mutant gene are also suitable for targeting. A sensestrand is designed based on the sequence of the selected portion.Further, siRNAs with lower G/C content (35-55%) may be more active thanthose with G/C content higher than 55%. Thus in one embodiment, theinvention includes nucleic acid molecules having 35-55% G/C content.

2. The sense strand of the siRNA is designed based on the sequence ofthe selected target site. Preferably the sense strand includes about 19to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. Morepreferably, the sense strand includes 21, 22 or 23 nucleotides. Theskilled artisan will appreciate, however, that siRNAs having a length ofless than 19 nucleotides or greater than 25 nucleotides can alsofunction to mediate RNAi. Accordingly, siRNAs of such length are alsowithin the scope of the instant invention provided that they retain theability to mediate RNAi. Longer RNA silencing agents have beendemonstrated to ellicit an interferon or PKR response in certainmammalian cells which may be undesirable. Preferably the RNA silencingagents of the invention do not ellicit a PKR response (i.e., are of asufficiently short length). However, longer RNA silencing agents may beuseful, for example, in cell types incapable of generating a PRKresponse or in situations where the PKR response has been downregulatedor dampened by alternative means.

The siRNA molecules of the invention have sufficient complementaritywith the target site such that the siRNA can mediate RNAi. In general,siRNA containing nucleotide sequences sufficiently identical to aportion of the target gene to effect RISC-mediated cleavage of thetarget gene are preferred. Accordingly, in a preferred embodiment, thesense strand of the siRNA is designed have to have a sequencesufficiently identical to a portion of the target. For example, thesense strand may have 100% identity to the target site. However, 100%identity is not required. Greater than 80% identity, e.g., 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or even 100% identity, between the sense strand andthe target RNA sequence is preferred. The invention has the advantage ofbeing able to tolerate certain sequence variations to enhance efficiencyand specificity of RNAi. In one embodiment, the sense strand has 4, 3,2, 1, or 0 mismatched nucleotide(s) with a target region, such as atarget region that differs by at least one base pair between the wildtype and mutant allele, e.g., a target region comprising thegain-of-function mutation, and the other strand is identical orsubstantially identical to the first strand. Moreover, siRNA sequenceswith small insertions or deletions of 1 or 2 nucleotides may also beeffective for mediating RNAi. Alternatively, siRNA sequences withnucleotide analog substitutions or insertions can be effective forinhibition.

Sequence identity may determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

3. siRNAs are designed such that perfect complementarity exists betweenthe siRNA and the target mRNA (e.g., the mutant mRNA) at thepolymorphism (e.g., the point mutation), there thus being a mismatch ifthe siRNA is compared (e.g., aligned) to the reference sequence (e.g.,wild type allele or mRNA sequence). The sense strand sequence may bedesigned such that the polymorphism is essentially in the middle of thestrand. For example, if a 21-nucleotide siRNA is chosen, thepolymorphism is at, for example, nucleotide 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16 (i.e., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotidesfrom the 5′ end of the sense strand. For a 22-nucleotide siRNA, thepolymorphism is at, for example, nucleotide 7, 8, 9, 10, 11, 12, 13, 14,15 or 16. For a 23-nucleotide siRNA, the polymorphism is at, forexample, 7, 8, 9, 10, 11, 12, 13, 14, or 16. For a 24-nucleotide siRNA,the polymorphism is at, for example, 9, 10, 11, 12, 13, 14 or 16. For a25-nucleotide siRNA, the polymorphism is at, for example, 9, 10, 11, 12,13, 14, 15, 16 or 17.

In one preferred embodiment, the sense strand of the siRNA is identicalto the polymorphism at a nucleotide position that is 10 nucleotides fromthe 5′ end of the sense strand (i.e., position P10).

In another preferred embodiment, the sense strand of the siRNA isidentical to the polymorphism at a nucleotide position that is 16nucleotides from the 5′ end of the sense strand (i.e., position P16).

4. siRNAs with single nucleotide specificity are preferably designedsuch that base paring at the single nucleotide in the correspondingreference (e.g., wild type) sequence is disfavored. For example,designing the siRNA such that purine:purine paring exists between thesiRNA and the wild type mRNA at the single nucleotide enhances singlenucleotide specificity. The purine:purine paring is selected, forexample, from the group G:G, A:G, G:A and A:A pairing. Moreover, purinepyrimidine pairing between the siRNA and the mutant mRNA at the singlenucleotide enhances single nucleotide specificity. The purine:pyrimidineparing is selected, for example, from the group G:C, C:G, A:U, U:A, C:A,A:C, U:A and A:U pairing.

5. The antisense or guide strand of the siRNA is routinely the samelength as the sense strand and includes complementary nucleotides. Inone embodiment, the guide and sense strands are fully complementary,i.e., the strands are blunt-ended when aligned or annealed. In anotherembodiment, the strands of the siRNA can be paired in such a way as tohave a 3′ overhang of 1 to 4, e.g., 2, nucleotides. Overhangs cancomprise (or consist of) nucleotides corresponding to the target genesequence (or complement thereof). Alternatively, overhangs can comprise(or consist of) deoxyribonucleotides, for example dTs, or nucleotideanalogs, or other suitable non-nucleotide material. Thus in anotherembodiment, the nucleic acid molecules may have a 3′ overhang of 2nucleotides, such as TT. The overhanging nucleotides may be either RNAor DNA. As noted above, it is desirable to choose a target regionwherein the mutant:wild type mismatch is a purine:purine mismatch.

6. Using any method known in the art, compare the potential targets tothe appropriate genome database (human, mouse, rat, etc.) and eliminatefrom consideration any target sequences with significant homology toother coding sequences. One such method for such sequence homologysearches is known as BLAST, which is available at National Center forBiotechnology Information website.

7. Select one or more sequences that meet your criteria for evaluation.

Further general information about the design and use of siRNA may befound in “The siRNA User Guide,” available at The Max-Plank-Institut fürBiophysikalishe Chemie website.

Alternatively, the siRNA may be defined functionally as a nucleotidesequence (or oligonucleotide sequence) that is capable of hybridizingwith the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed bywashing). Additional preferred hybridization conditions includehybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamidefollowed by washing at 70° C. in 0.3×SSC or hybridization at 70° C. in4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in1×SSC. The hybridization temperature for hybrids anticipated to be lessthan 50 base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference.

Negative control siRNAs should have the same nucleotide composition asthe selected siRNA, but without significant sequence complementarity tothe appropriate genome. Such negative controls may be designed byrandomly scrambling the nucleotide sequence of the selected siRNA; ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence.

8. To validate the effectiveness by which siRNAs destroy mutant mRNAs(e.g., mutant huntingtin mRNA), the siRNA may be incubated with mutantcDNA (e.g., mutant huntingtin cDNA) in a Drosophila-based in vitro mRNAexpression system. Radiolabeled with ³²P, newly synthesized mutant mRNAs(e.g., mutant huntingtin mRNA) are detected autoradiographically on anagarose gel. The presence of cleaved mutant mRNA indicates mRNA nucleaseactivity. Suitable controls include omission of siRNA and use ofwild-type huntingtin cDNA. Alternatively, control siRNAs are selectedhaving the same nucleotide composition as the selected siRNA, butwithout significant sequence complementarity to the appropriate targetgene. Such negative controls can be designed by randomly scrambling thenucleotide sequence of the selected siRNA; a homology search can beperformed to ensure that the negative control lacks homology to anyother gene in the appropriate genome. In addition, negative controlsiRNAs can be designed by introducing one or more base mismatches intothe sequence.

Anti-htt siRNAs may be designed to target any of the single nucleotidepolymorphisms described supra. Said siRNAs comprise an antisense strandwhich is fully complementary with the single nucleotide polymorphism. Incertain embodiments, the RNA silencing agent is a siRNA.

In certain embodiments, the siRNA comprises (i) a sense strandcomprising the sequence set forth as SEQ ID NO: 34; and (ii) anantisense strand comprising the sequence set forth as SEQ ID NO: 35. Inanother embodiment, the siRNA comprises (i) a sense strand comprisingthe sequence set forth as SEQ ID NO: 38; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO: 39. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO: 40; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO: 41. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO: 44; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO: 45. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO: 46; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO: 47. In anotherembodiment, the siRNA comprises (i) a sense strand comprising thesequence set forth as SEQ ID NO: 49; and (ii) an antisense strandcomprising the sequence set forth as SEQ ID NO: 50.

To validate the effectiveness by which siRNAs destroy mutant mRNAs(e.g., mutant huntingtin mRNA), the siRNA is incubated with mutant cDNA(e.g., mutant huntingtin cDNA) in a Drosophila-based in vitro mRNAexpression system. Radiolabeled with ³²P, newly synthesized mutant mRNAs(e.g., mutant huntingtin mRNA) are detected autoradiographically on anagarose gel. The presence of cleaved mutant mRNA indicates mRNA nucleaseactivity. Suitable controls include omission of siRNA and use ofwild-type huntingtin cDNA. Alternatively, control siRNAs are selectedhaving the same nucleotide composition as the selected siRNA, butwithout significant sequence complementarity to the appropriate targetgene. Such negative controls can be designed by randomly scrambling thenucleotide sequence of the selected siRNA; a homology search can beperformed to ensure that the negative control lacks homology to anyother gene in the appropriate genome. In addition, negative controlsiRNAs can be designed by introducing one or more base mismatches intothe sequence.

Sites of siRNA-mRNA complementation are selected which result in optimalmRNA specificity and maximal mRNA cleavage.

While the instant invention primarily features targeting polymorphicregions in the target mutant gene (e.g., in mutant htt) distinct fromthe expanded CAG region mutation, the skilled artisan will appreciatethat targeting the mutant region may have applicability as a therapeuticstrategy in certain situations. Targeting the mutant region can beaccomplished using siRNA that complements CAG in series. The siRNA^(cag)would bind to mRNAs with CAG complementation, but might be expected tohave greater opportunity to bind to an extended CAG series. MultiplesiRNA^(cag) would bind to the mutant huntingtin mRNA (as opposed tofewer for the wild type huntingtin mRNA); thus, the mutant huntingtinmRNA is more likely to be cleaved. Successful mRNA inactivation usingthis approach would also eliminate normal or wild-type huntingtin mRNA.Also inactivated, at least to some extent, could be other normal genes(approximately 70) which also have CAG repeats, where their mRNAs couldinteract with the siRNA. This approach would thus rely on an attritionstrategy—more of the mutant huntingtin mRNA would be destroyed than wildtype huntingtin mRNA or the other approximately 69 mRNAs that code forpolyglutamines.

b) siRNA-Like Molecules

siRNA-like molecules of the invention have a sequence (i.e., have astrand having a sequence) that is “sufficiently complementary” to aheterozygous SNP of a htt mRNA to direct gene silencing either by RNAior translational repression. siRNA-like molecules are designed in thesame way as siRNA molecules, but the degree of sequence identity betweenthe sense strand and target RNA approximates that observed between anmiRNA and its target. In general, as the degree of sequence identitybetween a miRNA sequence and the corresponding target gene sequence isdecreased, the tendency to mediate post-transcriptional gene silencingby translational repression rather than RNAi is increased. Therefore, inan alternative embodiment, where post-transcriptional gene silencing bytranslational repression of the target gene is desired, the miRNAsequence has partial complementarity with the target gene sequence. Incertain embodiments, the miRNA sequence has partial complementarity withone or more short sequences (complementarity sites) dispersed within thetarget mRNA (e.g. within the 3′-UTR of the target mRNA) (Hutvagner andZamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA,2003; Doench et al., Genes & Dev., 2003). Since the mechanism oftranslational repression is cooperative, multiple complementarity sites(e.g., 2, 3, 4, 5, or 6) may be targeted in certain embodiments.

The capacity of a siRNA-like duplex to mediate RNAi or translationalrepression may be predicted by the distribution of non-identicalnucleotides between the target gene sequence and the nucleotide sequenceof the silencing agent at the site of complementarity. In oneembodiment, where gene silencing by translational repression is desired,at least one non-identical nucleotide is present in the central portionof the complementarity site so that duplex formed by the miRNA guidestrand and the target mRNA contains a central “bulge” (Doench J G etal., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5, or 6contiguous or non-contiguous non-identical nucleotides are introduced.The non-identical nucleotide may be selected such that it forms a wobblebase pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G,A:A, C:C, U:U). In a further preferred embodiment, the “bulge” iscentered at nucleotide positions 12 and 13 from the 5′ end of the miRNAmolecule.

c) Short Hairpin RNA (shRNA) Molecules

In certain featured embodiments, the instant invention provides shRNAscapable of mediating RNA silencing of a heterozygous htt SNP withenhanced selectivity. In contrast to siRNAs, shRNAs mimic the naturalprecursors of micro RNAs (miRNAs) and enter at the top of the genesilencing pathway. For this reason, shRNAs are believed to mediate genesilencing more efficiently by being fed through the entire natural genesilencing pathway.

miRNAs are noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel during plant and animal development. One common feature of miRNAsis that they are all excised from an approximately 70 nucleotideprecursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNaseIII-type enzyme, or a homolog thereof. Naturally-occurring miRNAprecursors (pre-miRNA) have a single strand that forms a duplex stemincluding two portions that are generally complementary, and a loop,that connects the two portions of the stem. In typical pre-miRNAs, thestem includes one or more bulges, e.g., extra nucleotides that create asingle nucleotide “loop” in one portion of the stem, and/or one or moreunpaired nucleotides that create a gap in the hybridization of the twoportions of the stem to each other. Short hairpin RNAs, or engineeredRNA precursors, of the invention are artificial constructs based onthese naturally occurring pre-miRNAs, but which are engineered todeliver desired RNA silencing agents (e.g., siRNAs of the invention). Bysubstituting the stem sequences of the pre-miRNA with sequencecomplementary to the target mRNA, a shRNA is formed. The shRNA isprocessed by the entire gene silencing pathway of the cell, therebyefficiently mediating RNAi.

The requisite elements of a shRNA molecule include a first portion and asecond portion, having sufficient complementarity to anneal or hybridizeto form a duplex or double-stranded stem portion. The two portions neednot be fully or perfectly complementary. The first and second “stem”portions are connected by a portion having a sequence that hasinsufficient sequence complementarity to anneal or hybridize to otherportions of the shRNA. This latter portion is referred to as a “loop”portion in the shRNA molecule. The shRNA molecules are processed togenerate siRNAs. shRNAs can also include one or more bulges, i.e., extranucleotides that create a small nucleotide “loop” in a portion of thestem, for example a one-, two- or three-nucleotide loop. The stemportions can be the same length, or one portion can include an overhangof, for example, 1-5 nucleotides. The overhanging nucleotides caninclude, for example, uracils (Us), e.g., all Us. Such Us are notablyencoded by thymidines (Ts) in the shRNA-encoding DNA which signal thetermination of transcription.

In shRNAs, or engineered precursor RNAs, of the instant invention, oneportion of the duplex stem is a nucleic acid sequence that iscomplementary (or anti-sense) to the heterozygous SNP. Preferably, onestrand of the stem portion of the shRNA is sufficiently complementary(e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediatedegradation or cleavage of said target RNA via RNA interference (RNAi).Thus, engineered RNA precursors include a duplex stem with two portionsand a loop connecting the two stem portions. The antisense portion canbe on the 5′ or 3′ end of the stem. The stem portions of a shRNA arepreferably about 15 to about 50 nucleotides in length. Preferably thetwo stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30,35, 37, 38, 39, or 40 or more nucleotides in length. In preferredembodiments, the length of the stem portions should be 21 nucleotides orgreater. When used in mammalian cells, the length of the stem portionsshould be less than about 30 nucleotides to avoid provoking non-specificresponses like the interferon pathway. In non-mammalian cells, the stemcan be longer than 30 nucleotides. In fact, the stem can include muchlarger sections complementary to the target mRNA (up to, and includingthe entire mRNA). In fact, a stem portion can include much largersections complementary to the target mRNA (up to, and including theentire mRNA).

The two portions of the duplex stem must be sufficiently complementaryto hybridize to form the duplex stem. Thus, the two portions can be, butneed not be, fully or perfectly complementary. In addition, the two stemportions can be the same length, or one portion can include an overhangof 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include,for example, uracils (Us), e.g., all Us. The loop in the shRNAs orengineered RNA precursors may differ from natural pre-miRNA sequences bymodifying the loop sequence to increase or decrease the number of pairednucleotides, or replacing all or part of the loop sequence with atetraloop or other loop sequences. Thus, the loop in the shRNAs orengineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g.,15 or 20, or more nucleotides in length.

The loop in the shRNAs or engineered RNA precursors may differ fromnatural pre-miRNA sequences by modifying the loop sequence to increaseor decrease the number of paired nucleotides, or replacing all or partof the loop sequence with a tetraloop or other loop sequences. Thus, theloop portion in the shRNA can be about 2 to about 20 nucleotides inlength, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, ormore nucleotides in length. A preferred loop consists of or comprises a“tetraloop” sequences. Exemplary tetraloop sequences include, but arenot limited to, the sequences GNRA, where N is any nucleotide and R is apurine nucleotide, GGGG, and UUUU.

In certain embodiments, shRNAs of the invention include the sequences ofa desired siRNA molecule described supra. In other embodiments, thesequence of the antisense portion of a shRNA can be designed essentiallyas described above or generally by selecting an 18, 19, 20, 21nucleotide, or longer, sequence from within the target RNA (e.g., SOD1or htt mRNA), for example, from a region 100 to 200 or 300 nucleotidesupstream or downstream of the start of translation. In general, thesequence can be selected from any portion of the target RNA (e.g., mRNA)including the 5′ UTR (untranslated region), coding sequence, or 3′ UTR,provided said portion is distant from the site of the gain-of-functionmutation. This sequence can optionally follow immediately after a regionof the target gene containing two adjacent AA nucleotides. The last twonucleotides of the nucleotide sequence can be selected to be UU. This 21or so nucleotide sequence is used to create one portion of a duplex stemin the shRNA. This sequence can replace a stem portion of a wild-typepre-miRNA sequence, e.g., enzymatically, or is included in a completesequence that is synthesized. For example, one can synthesize DNAoligonucleotides that encode the entire stem-loop engineered RNAprecursor, or that encode just the portion to be inserted into theduplex stem of the precursor, and using restriction enzymes to build theengineered RNA precursor construct, e.g., from a wild-type pre-miRNA.

Engineered RNA precursors include in the duplex stem the 21-22 or sonucleotide sequences of the siRNA or siRNA-like duplex desired to beproduced in vivo. Thus, the stem portion of the engineered RNA precursorincludes at least 18 or 19 nucleotide pairs corresponding to thesequence of an exonic portion of the gene whose expression is to bereduced or inhibited. The two 3′ nucleotides flanking this region of thestem are chosen so as to maximize the production of the siRNA from theengineered RNA precursor and to maximize the efficacy of the resultingsiRNA in targeting the corresponding mRNA for translational repressionor destruction by RNAi in vivo and in vitro.

In certain embodiments, shRNAs of the invention include miRNA sequences,optionally end-modified miRNA sequences, to enhance entry into RISC. ThemiRNA sequence can be similar or identical to that of any naturallyoccurring miRNA (see e.g. The miRNA Registry; Griffiths-Jones S, Nuc.Acids Res., 2004). Over one thousand natural miRNAs have been identifiedto date and together they are thought to comprise ˜1% of all predictedgenes in the genome. Many natural miRNAs are clustered together in theintrons of pre-mRNAs and can be identified in silico usinghomology-based searches (Pasquinelli et al., 2000; Lagos-Quintana etal., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computeralgorithms (e.g. MiRScan, MiRSeeker) that predict the capability of acandidate miRNA gene to form the stem loop structure of a pri-mRNA (Gradet al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al.,Science, 2003; Lai E C et al., Genome Bio., 2003). An online registryprovides a searchable database of all published miRNA sequences (ThemiRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc.Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7,miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs,as well as other natural miRNAs from humans and certain model organismsincluding Drosophila melanogaster, Caenorhabditis elegans, zebrafish,Arabidopsis thalania, mouse, and rat as described in International PCTPublication No. WO 03/029459.

Naturally-occurring miRNAs are expressed by endogenous genes in vivo andare processed from a hairpin or stem-loop precursor (pre-miRNA orpri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science,2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001;Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev.,2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003;Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al.,Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can existtransiently in vivo as a double-stranded duplex but only one strand istaken up by the RISC complex to direct gene silencing. Certain miRNAs,e.g. plant miRNAs, have perfect or near-perfect complementarity to theirtarget mRNAs and, hence, direct cleavage of the target mRNAs. OthermiRNAs have less than perfect complementarity to their target mRNAs and,hence, direct translational repression of the target mRNAs. The degreeof complementarity between an miRNA and its target mRNA is believed todetermine its mechanism of action. For example, perfect or near-perfectcomplementarity between a miRNA and its target mRNA is predictive of acleavage mechanism (Yekta et al., Science, 2004), whereas less thanperfect complementarity is predictive of a translational repressionmechanism. In particular embodiments, the miRNA sequence is that of anaturally-occurring miRNA sequence, the aberrant expression or activityof which is correlated with a miRNA disorder.

d) Dual Functional Oligonucleotide Tethers

In other embodiments, the RNA silencing agents of the present inventioninclude dual functional oligonucleotide tethers useful for theintercellular recruitment of a miRNA. Animal cells express a range ofmiRNAs, noncoding RNAs of approximately 22 nucleotides which canregulate gene expression at the post transcriptional or translationallevel. By binding a miRNA bound to RISC and recruiting it to a targetmRNA, a dual functional oligonucleotide tether can repress theexpression of genes involved e.g., in the arteriosclerotic process. Theuse of oligonucleotide tethers offer several advantages over existingtechniques to repress the expression of a particular gene. First, themethods described herein allow an endogenous molecule (often present inabundance), an miRNA, to mediate RNA silencing; accordingly the methodsdescribed herein obviate the need to introduce foreign molecules (e.g.,siRNAs) to mediate RNA silencing. Second, the RNA-silencing agents and,in particular, the linking moiety (e.g., oligonucleotides such as the2′-O-methyl oligonucleotide), can be made stable and resistant tonuclease activity. As a result, the tethers of the present invention canbe designed for direct delivery, obviating the need for indirectdelivery (e.g. viral) of a precursor molecule or plasmid designed tomake the desired agent within the cell. Third, tethers and theirrespective moieties, can be designed to conform to specific mRNA sitesand specific miRNAs. The designs can be cell and gene product specific.Fourth, the methods disclosed herein leave the mRNA intact, allowing oneskilled in the art to block protein synthesis in short pulses using thecell's own machinery. As a result, these methods of RNA silencing arehighly regulatable.

The dual functional oligonucleotide tethers (“tethers”) of the inventionare designed such that they recruit miRNAs (e.g., endogenous cellularmiRNAs) to a target mRNA so as to induce the modulation of a gene ofinterest. In preferred embodiments, the tethers have the formula T-L-μ,wherein T is an mRNA targeting moiety, L is a linking moiety, and μ isan miRNA recruiting moiety. Any one or more moiety may be doublestranded. Preferably, however, each moiety is single stranded.

Moieties within the tethers can be arranged or linked (in the 5′ to 3′direction) as depicted in the formula T-L-μ (i.e., the 3′ end of thetargeting moiety linked to the 5′ end of the linking moiety and the 3′end of the linking moiety linked to the 5′ end of the miRNA recruitingmoiety). Alternatively, the moieties can be arranged or linked in thetether as follows: μ-T-L (i.e., the 3′ end of the miRNA recruitingmoiety linked to the 5′ end of the linking moiety and the 3′ end of thelinking moiety linked to the 5′ end of the targeting moiety).

The mRNA targeting moiety, as described above, is capable of capturing aspecific target mRNA. According to the invention, expression of thetarget mRNA is undesirable, and, thus, translational repression of themRNA is desired. The mRNA targeting moiety should be of sufficient sizeto effectively bind the target mRNA. The length of the targeting moietywill vary greatly depending, in part, on the length of the target mRNAand the degree of complementarity between the target mRNA and thetargeting moiety. In various embodiments, the targeting moiety is lessthan about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, or 5 nucleotides in length. In a particular embodiment,the targeting moiety is about 15 to about 25 nucleotides in length.

The miRNA recruiting moiety, as described above, is capable ofassociating with a miRNA. According to the invention, the miRNA may beany miRNA capable of repressing the target mRNA. Mammals are reported tohave over 250 endogenous miRNAs (Lagos-Quintana et al. (2002) CurrentBiol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; andLim et al. (2003) Science 299:1540). In various embodiments, the miRNAmay be any art-recognized miRNA. Table 3 lists some of the known humanmiRNAs

The linking moiety is any agent capable of linking the targetingmoieties such that the activity of the targeting moieties is maintained.Linking moieties are preferably oligonucleotide moieties comprising asufficient number of nucleotides such that the targeting agents cansufficiently interact with their respective targets. Linking moietieshave little or no sequence homology with cellular mRNA or miRNAsequences. Exemplary linking moieties include one or more 2′-O—methylnucleotides, e.g., 2′-β-methyladenosine, 2′-O-methylthymidine,2′-O-methylguanosine or 2′-O-methyluridine.

VII. Modified Anti-Htt RNA Silencing Agents

In certain aspects of the invention, an RNA silencing agent (or anyportion thereof) of the invention as described supra may be modifiedsuch that the activity of the agent is further improved. For example,the RNA silencing agents described in Section II supra may be modifiedwith any of the modifications described infra. The modifications can, inpart, serve to further enhance target discrimination, to enhancestability of the agent (e.g., to prevent degradation), to promotecellular uptake, to enhance the target efficiency, to improve efficacyin binding (e.g., to the targets), to improve patient tolerance to theagent, and/or to reduce toxicity.

1) Modifications to Enhance Target Discrimination

In certain embodiments, the RNA silencing agents of the invention may besubstituted with a destabilizing nucleotide to enhance single nucleotidetarget discrimination (see U.S. application Ser. No. 11/698,689, filedJan. 25, 2007 and U.S. Provisional Application No. 60/762,225 filed Jan.25, 2006, both of which are incorporated herein by reference). Such amodification may be sufficient to abolish the specificity of the RNAsilencing agent for a non-target mRNA (e.g. wild-type mRNA), withoutappreciably affecting the specificity of the RNA silencing agent for atarget mRNA (e.g. gain-of-function mutant mRNA).

In preferred embodiments, the RNA silencing agents of the invention aremodified by the introduction of at least one universal nucleotide in theantisense strand thereof. Universal nucleotides comprise base portionsthat are capable of base pairing indiscriminately with any of the fourconventional nucleotide bases (e.g. A,G,C,U). A universal nucleotide ispreferred because it has relatively minor effect on the stability of theRNA duplex or the duplex formed by the guide strand of the RNA silencingagent and the target mRNA. Exemplary universal nucleotide include thosehaving an inosine base portion or an inosine analog base portionselected from the group consisting of deoxyinosine (e.g.2′-deoxyinosine), 7-deaza-2′-deoxyinosine, 2′-aza-2′-deoxyinosine,PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine,2′-O-methoxyethyl-inosine, and 2′-OMe-inosine. In particularly preferredembodiments, the universal nucleotide is an inosine residue or anaturally occurring analog thereof.

The RNA silencing agents of the invention are preferably modified by theintroduction of at least one destabilizing nucleotide within 5nucleotides from a specificity-determining nucleotide (ie. thenucleotide which recognizes the disease-related polymorphism). Forexample, the destabilizing nucleotide may be introduced at a positionthat is within 5, 4, 3, 2, or 1 nucleotide(s) from aspecificity-determining nucleotide. In exemplary embodiments, thedestabilizing nucleotide is introduced at a position which is 3nucleotides from the specificity-determining nucleotide (ie. such thatthere are 2 stabilizing nucleotides between the destablilizingnucleotide and the specificity-determining nucleotide). In RNA silencingagents having two strands or strand portions (e.g. siRNAs and shRNAs),the destabilizing nucleotide may be introduced in the strand or strandportion that does not contain the specificity-determining nucleotide. Inpreferred embodiments, the destabilizing nucleotide is introduced in thesame strand or strand portion that contains the specificity-determiningnucleotide.

2) Modifications to Enhance Efficacy and Specificity

In certain embodiments, the RNA silencing agents of the invention may bealtered to facilitate enhanced efficacy and specificity in mediatingRNAi according to asymmetry design rules (see International PublicationNo. WO 2005/001045, US Publication No. 2005-0181382 A1). Suchalterations facilitate entry of the antisense strand of the siRNA (e.g.,a siRNA designed using the methods of the invention or an siRNA producedfrom a shRNA) into RISC in favor of the sense strand, such that theantisense strand preferentially guides cleavage or translationalrepression of a target mRNA, and thus increasing or improving theefficiency of target cleavage and silencing. Preferably the asymmetry ofan RNA silencing agent is enhanced by lessening the base pair strengthbetween the antisense strand 5′ end (AS 5′) and the sense strand 3′ end(S 3′) of the RNA silencing agent relative to the bond strength or basepair strength between the antisense strand 3′ end (AS 3′) and the sensestrand 5′ end (S ′5) of said RNA silencing agent.

In one embodiment, the asymmetry of an RNA silencing agent of theinvention may be enhanced such that there are fewer G:C base pairsbetween the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion than between the 3′ end of the first orantisense strand and the 5′ end of the sense strand portion. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one mismatched base pair betweenthe 5′ end of the first or antisense strand and the 3′ end of the sensestrand portion. Preferably, the mismatched base pair is selected fromthe group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In anotherembodiment, the asymmetry of an RNA silencing agent of the invention maybe enhanced such that there is at least one wobble base pair, e.g., G:U,between the 5′ end of the first or antisense strand and the 3′ end ofthe sense strand portion. In another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a rare nucleotide, e.g., inosine (I).Preferably, the base pair is selected from the group consisting of anI:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNAsilencing agent of the invention may be enhanced such that there is atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

3) RNA Silencing Agents with Enhanced Stability

The RNA silencing agents of the present invention can be modified toimprove stability in serum or in growth medium for cell cultures. Inorder to enhance the stability, the 3′-residues may be stabilizedagainst degradation, e.g., they may be selected such that they consistof purine nucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a preferred aspect, the invention features RNA silencing agents thatinclude first and second strands wherein the second strand and/or firststrand is modified by the substitution of internal nucleotides withmodified nucleotides, such that in vivo stability is enhanced ascompared to a corresponding unmodified RNA silencing agent. As definedherein, an “internal” nucleotide is one occurring at any position otherthan the 5′ end or 3′ end of nucleic acid molecule, polynucleotide oroligonucleotide. An internal nucleotide can be within a single-strandedmolecule or within a strand of a duplex or double-stranded molecule. Inone embodiment, the sense strand and/or antisense strand is modified bythe substitution of at least one internal nucleotide. In anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. Inanother embodiment, the sense strand and/or antisense strand is modifiedby the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of theinternal nucleotides. In yet another embodiment, the sense strand and/orantisense strand is modified by the substitution of all of the internalnucleotides.

In a preferred embodiment of the present invention the RNA silencingagents may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific silencing activity, e.g., the RNAi mediating activity ortranslational repression activity is not substantially effected, e.g.,in a region at the 5′-end and/or the 3′-end of the siRNA molecule.Particularly, the ends may be stabilized by incorporating modifiednucleotide analogues.

Preferred nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

In particular embodiments, the modifications are 2′-fluoro, 2′-aminoand/or 2′-thio modifications. Particularly preferred modificationsinclude 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine,2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine,2′-amino-adenosine, 2′-amino-guanosine, 2,6-diaminopurine,4-thio-uridine, and/or 5-amino-allyl-uridine. In a particularembodiment, the 2′-fluoro ribonucleotides are every uridine andcytidine. Additional exemplary modifications include 5-bromo-uridine,5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine,2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and5-fluoro-uridine. 2′-deoxy-nucleotides and 2′-Ome nucleotides can alsobe used within modified RNA-silencing agents moities of the instantinvention. Additional modified residues include, deoxy-abasic, inosine,N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purineribonucleoside and ribavirin. In a particularly preferred embodiment,the 2′ moiety is a methyl group such that the linking moiety is a2′-O-methyl oligonucleotide.

In an exemplary embodiment, the RNA silencing agent of the inventioncomprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modifiednucleotides that resist nuclease activities (are highly stable) andpossess single nucleotide discrimination for mRNA (Elmen et al., NucleicAcids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). Thesemolecules have 2′-O,4′-C-ethylene-bridged nucleic acids, with possiblemodifications such as 2′-deoxy-2″-fluorouridine. Moreover, LNAs increasethe specificity of oligonucleotides by constraining the sugar moietyinto the 3′-endo conformation, thereby preorganizing the nucleotide forbase pairing and increasing the melting temperature of theoligonucleotide by as much as 10° C. per base.

In another exemplary embodiment, the RNA silencing agent of theinvention comprises Peptide Nucleic Acids (PNAs). PNAs comprise modifiednucleotides in which the sugar-phosphate portion of the nucleotide isreplaced with a neutral 2-amino ethylglycine moiety capable of forming apolyamide backbone which is highly resistant to nuclease digestion andimparts improved binding specificity to the molecule (Nielsen, et al.,Science, (2001), 254: 1497-1500).

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

In other embodiments, cross-linking can be employed to alter thepharmacokinetics of the RNA silencing agent, for example, to increasehalf-life in the body. Thus, the invention includes RNA silencing agentshaving two complementary strands of nucleic acid, wherein the twostrands are crosslinked. The invention also includes RNA silencingagents which are conjugated or unconjugated (e.g., at its 3′ terminus)to another moiety (e.g. a non-nucleic acid moiety such as a peptide), anorganic compound (e.g., a dye), or the like). Modifying siRNAderivatives in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting siRNA derivative as compared tothe corresponding siRNA, are useful for tracing the siRNA derivative inthe cell, or improve the stability of the siRNA derivative compared tothe corresponding siRNA.

Other exemplary modifications include: (a) 2′ modification, e.g.,provision of a 2′ OMe moiety on a U in a sense or antisense strand, butespecially on a sense strand, or provision of a 2′ OMe moiety in a 3′overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom ofthe molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′position, as indicated by the context); (b) modification of thebackbone, e.g., with the replacement of an 0 with an S, in the phosphatebackbone, e.g., the provision of a phosphorothioate modification, on theU or the A or both, especially on an antisense strand; e.g., with thereplacement of a P with an S; (c) replacement of the U with a C5 aminolinker; (d) replacement of an A with a G (sequence changes are preferredto be located on the sense strand and not the antisense strand); and (d)modification at the 2′, 6′, 7′, or 8′ position. Preferred embodimentsare those in which one or more of these modifications are present on thesense but not the antisense strand, or embodiments where the antisensestrand has fewer of such modifications. Yet other exemplarymodifications include the use of a methylated P in a 3′ overhang, e.g.,at the 3′ terminus; combination of a 2′ modification, e.g., provision ofa 2′ O Me moiety and modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P, in a 3′ overhang, e.g., atthe 3′ terminus; modification with a 3′ alkyl; modification with anabasic pyrrolidine in a 3′ overhang, e.g., at the 3′ terminus;modification with naproxen, ibuprofen, or other moieties which inhibitdegradation at the 3′ terminus.

4) Modifications to Enhance Cellular Uptake

In other embodiments, RNA silencing agents may be modified with chemicalmoieties, for example, to enhance cellular uptake by target cells (e.g.,neuronal cells). Thus, the invention includes RNA silencing agents whichare conjugated or unconjugated (e.g., at its 3′ terminus) to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye), or the like. The conjugation can be accomplishedby methods known in the art, e.g., using the methods of Lambert et al.,Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loadedto polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J.Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound tonanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994)(describes nucleic acids linked to intercalating agents, hydrophobicgroups, polycations or PACA nanoparticles); and Godard et al., Eur. J.Biochem. 232(2):404-10 (1995) (describes nucleic acids linked tonanoparticles).

In a particular embodiment, an RNA silencing agent of invention isconjugated to a lipophilic moiety. In one embodiment, the lipophilicmoiety is a ligand that includes a cationic group. In anotherembodiment, the lipophilic moiety is attached to one or both strands ofan siRNA. In a preferred embodiment, the lipophilic moiety is attachedto one end of the sense strand of the siRNA. In another preferredembodiment, the lipophilic moiety is attached to the 3′ end of the sensestrand. In certain embodiments, the lipophilic moeity is selected fromthe group consisting of cholesterol, vitamin E, vitamin K, vitamin A,folic acid, or a cationic dye (e.g., Cy3). In a preferred embodiment,the lipophilic moiety is a cholesterol. Other lipophilic moietiesinclude cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

5) Tethered Ligands

Other entities can be tethered to an RNA silencing agent of theinvention. For example, a ligand tethered to an RNA silencing agent toimprove stability, hybridization thermodynamics with a target nucleicacid, targeting to a particular tissue or cell-type, or cellpermeability, e.g., by an endocytosis-dependent or -independentmechanism. Ligands and associated modifications can also increasesequence specificity and consequently decrease off-site targeting. Atethered ligand can include one or more modified bases or sugars thatcan function as intercalators. These are preferably located in aninternal region, such as in a bulge of RNA silencing agent/targetduplex. The intercalator can be an aromatic, e.g., a polycyclic aromaticor heterocyclic aromatic compound. A polycyclic intercalator can havestacking capabilities, and can include systems with 2, 3, or 4 fusedrings. The universal bases described herein can be included on a ligand.In one embodiment, the ligand can include a cleaving group thatcontributes to target gene inhibition by cleavage of the target nucleicacid. The cleaving group can be, for example, a bleomycin (e.g.,bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline(e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lystripeptide), or metal ion chelating group. The metal ion chelating groupcan include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II)2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, oracridine, which can promote the selective cleavage of target RNA at thesite of the bulge by free metal ions, such as Lu(III). In someembodiments, a peptide ligand can be tethered to a RNA silencing agentto promote cleavage of the target RNA, e.g., at the bulge region. Forexample, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) canbe conjugated to a peptide (e.g., by an amino acid derivative) topromote target RNA cleavage. A tethered ligand can be an aminoglycosideligand, which can cause an RNA silencing agent to have improvedhybridization properties or improved sequence specificity. Exemplaryaminoglycosides include glycosylated polylysine, galactosylatedpolylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugatesof aminoglycosides, such as Neo-N-acridine, Neo-S-acridine,Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of anacridine analog can increase sequence specificity. For example, neomycinB has a high affinity for RNA as compared to DNA, but lowsequence-specificity. An acridine analog, neo-5-acridine has anincreased affinity for the HIV Rev-response element (RRE). In someembodiments the guanidine analog (the guanidinoglycoside) of anaminoglycoside ligand is tethered to an RNA silencing agent. In aguanidinoglycoside, the amine group on the amino acid is exchanged for aguanidine group. Attachment of a guanidine analog can enhance cellpermeability of an RNA silencing agent. A tethered ligand can be apoly-arginine peptide, peptoid or peptidomimetic, which can enhance thecellular uptake of an oligonucleotide agent.

Preferred ligands are coupled, preferably covalently, either directly orindirectly via an intervening tether, to a ligand-conjugated carrier. Inpreferred embodiments, the ligand is attached to the carrier via anintervening tether. In preferred embodiments, a ligand alters thedistribution, targeting or lifetime of an RNA silencing agent into whichit is incorporated. In preferred embodiments a ligand provides anenhanced affinity for a selected target, e.g, molecule, cell or celltype, compartment, e.g., a cellular or organ compartment, tissue, organor region of the body, as, e.g., compared to a species absent such aligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified RNA silencing agent, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides. Ligands in general can include therapeuticmodifiers, e.g., for enhancing uptake; diagnostic compounds or reportergroups e.g., for monitoring distribution; cross-linking agents;nuclease-resistance conferring moieties; and natural or unusualnucleobases. General examples include lipophiles, lipids, steroids(e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g.,sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid),vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal),carbohydrates, proteins, protein binding agents, integrin targetingmolecules, polycationics, peptides, polyamines, and peptide mimics.Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine, multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic.Other examples of ligands include dyes, intercalating agents (e.g.acridines and substituted acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine,phenanthroline, pyrenes), lys-tyr-lys tripeptide, aminoglycosides,guanidium aminoglycodies, artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol (and thio analogs thereof),cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid,1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters(e.g., mono, bis, or tris fatty acid esters, e.g., C₁₀, C₁₁, C₁₂, C₁₃,C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ fatty acids) and ethers thereof,e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ alkyl;e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octaadecyl)glycerol),geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g.,glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholicacid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) andpeptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylatingagents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g.,imidazole, bisimidazole, histamine, imidazole clusters,acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles),dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the RNA silencing agent into the cell, for example, bydisrupting the cell's cytoskeleton, e.g., by disrupting the cell'smicrotubules, microfilaments, and/or intermediate filaments. The drugcan be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin. The ligand can increase the uptake of the RNAsilencing agent into the cell by activating an inflammatory response,for example. Exemplary ligands that would have such an effect includetumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gammainterferon. In one aspect, the ligand is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, neproxin oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA. A lipid based ligand canbe used to modulate, e.g., control the binding of the conjugate to atarget tissue. For example, a lipid or lipid-based ligand that binds toHSA more strongly will be less likely to be targeted to the kidney andtherefore less likely to be cleared from the body. A lipid orlipid-based ligand that binds to HSA less strongly can be used to targetthe conjugate to the kidney. In a preferred embodiment, the lipid basedligand binds HSA. A lipid-based ligand can bind HSA with a sufficientaffinity such that the conjugate will be preferably distributed to anon-kidney tissue. However, it is preferred that the affinity not be sostrong that the HSA-ligand binding cannot be reversed. In anotherpreferred embodiment, the lipid based ligand binds HSA weakly or not atall, such that the conjugate will be preferably distributed to thekidney. Other moieties that target to kidney cells can also be used inplace of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics tooligonucleotide agents can affect pharmacokinetic distribution of theRNA silencing agent, such as by enhancing cellular recognition andabsorption. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long. A peptide or peptidomimetic can be, for example, a cellpermeation peptide, cationic peptide, amphipathic peptide, orhydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). Thepeptide moiety can be a dendrimer peptide, constrained peptide orcrosslinked peptide. The peptide moiety can be an L-peptide orD-peptide. In another alternative, the peptide moiety can include ahydrophobic membrane translocation sequence (MTS). A peptide orpeptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan RNA silencing agent via an incorporated monomer unit is a celltargeting peptide such as an arginine-glycine-aspartic acid(RGD)-peptide, or RGD mimic. A peptide moiety can range in length fromabout 5 amino acids to about 40 amino acids. The peptide moieties canhave a structural modification, such as to increase stability or directconformational properties. Any of the structural modifications describedbelow can be utilized.

VIII. Methods of Introducing Nucleic Acids, Vectors, and Host Cells

RNA silencing agents of the invention may be directly introduced intothe cell (e.g., a neural cell) (i.e., intracellularly); or introducedextracellularly into a cavity, interstitial space, into the circulationof an organism, introduced orally, or may be introduced by bathing acell or organism in a solution containing the nucleic acid. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the nucleic acid may be introduced.

The RNA silencing agents of the invention can be introduced usingnucleic acid delivery methods known in art including injection of asolution containing the nucleic acid, bombardment by particles coveredby the nucleic acid, soaking the cell or organism in a solution of thenucleic acid, or electroporation of cell membranes in the presence ofthe nucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or other-wiseincrease inhibition of the target gene.

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of gene expression refers to theabsence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS).

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of RNAi agent may result in inhibitionin a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%,or 95% of targeted cells). Quantization of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell; mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

In a preferred aspect, the efficacy of an RNAi agent of the invention(e.g., an siRNA targeting a polymorphism in a mutant gene) is tested forits ability to specifically degrade mutant mRNA (e.g., mutant htt mRNAand/or the production of mutant huntingtin protein) in cells, inparticular, in neurons (e.g., striatal or cortical neuronal clonal linesand/or primary neurons). Also suitable for cell-based validation assaysare other readily transfectable cells, for example, HeLa cells or COScells. Cells are transfected with human wild type or mutant cDNAs (e.g.,human wild type or mutant huntingtin cDNA). Standard siRNA, modifiedsiRNA or vectors able to produce siRNA from U-looped mRNA areco-transfected. Selective reduction in mutant mRNA (e.g., mutanthuntingtin mRNA) and/or mutant protein (e.g., mutant huntingtin) ismeasured. Reduction of mutant mRNA or protein can be compared to levelsof normal mRNA or protein. Exogenously-introduced normal mRNA or protein(or endogenous normal mRNA or protein) can be assayed for comparisonpurposes. When utilizing neuronal cells, which are known to be somewhatresistant to standard transfection techniques, it may be desirable tointroduce RNAi agents (e.g., siRNAs) by passive uptake.

IX. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by a gain of function mutantprotein. In one embodiment, the disease or disorder is a trinucleotiderepeat disease or disorder. In another embodiment, the disease ordisorder is a polyglutamine disorder. In a preferred embodiment, thedisease or disorder is a disorder associated with the expression ofhuntingtin and in which alteration of huntingtin, especially theamplification of CAG repeat copy number, leads to a defect in huntingtingene (structure or function) or huntingtin protein (structure orfunction or expression), such that clinical manifestations include thoseseen in Huntington's disease patients.

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., a RNA agent or vector ortransgene encoding same) to a patient, or application or administrationof a therapeutic agent to an isolated tissue or cell line from apatient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above, by administering tothe subject a therapeutic agent (e.g., an RNAi agent or vector ortransgene encoding same). Subjects at risk for the disease can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe disease or disorder, such that the disease or disorder is preventedor, alternatively, delayed in its progression.

Another aspect of the invention pertains to methods treating subjectstherapeutically, i.e., alter onset of symptoms of the disease ordisorder. In an exemplary embodiment, the modulatory method of theinvention involves contacting a cell expressing a gain-of-functionmutant with a therapeutic agent (e.g., a RNAi agent or vector ortransgene encoding same) that is specific for a polymorphism within thegene, such that sequence specific interference with the gene isachieved. These methods can be performed in vitro (e.g., by culturingthe cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject).

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

Therapeutic agents can be tested in an appropriate animal model. Forexample, an RNAi agent (or expression vector or transgene encoding same)as described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

A pharmaceutical composition containing an RNA silencing agent of theinvention can be administered to any patient diagnosed as having or atrisk for developing a neurological disorder, such as HD. In oneembodiment, the patient is diagnosed as having a neurological disorder,and the patient is otherwise in general good health. For example, thepatient is not terminally ill, and the patient is likely to live atleast 2, 3, 5, or years or longer following diagnosis. The patient canbe treated immediately following diagnosis, or treatment can be delayeduntil the patient is experiencing more debilitating symptoms, such asmotor fluctuations and dyskinesis in PD patients. In another embodiment,the patient has not reached an advanced stage of the disease.

An RNA silencing agent modified for enhance uptake into neural cells canbe administered at a unit dose less than about 1.4 mg per kg ofbodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005,0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, andless than 200 nmole of RNA agent (e.g., about 4.4×10¹⁶ copies) per kg ofbodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75,0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNAsilencing agent per kg of bodyweight. The unit dose, for example, can beadministered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into the brain), an inhaled dose, or atopical application. Particularly preferred dosages are less than 2, 1,or 0.1 mg/kg of body weight.

Delivery of an RNA silencing agent directly to an organ (e.g., directlyto the brain) can be at a dosage on the order of about 0.00001 mg toabout 3 mg per organ, or preferably about 0.0001-0.001 mg per organ,about 0.03-3.0 mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0mg per organ. The dosage can be an amount effective to treat or preventa neurological disease or disorder, e.g., HD. In one embodiment, theunit dose is administered less frequently than once a day, e.g., lessthan every 2, 4, 8 or 30 days. In another embodiment, the unit dose isnot administered with a frequency (e.g., not a regular frequency). Forexample, the unit dose may be administered a single time. In oneembodiment, the effective dose is administered with other traditionaltherapeutic modalities.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an RNA silencing agent. The maintenance doseor doses are generally lower than the initial dose, e.g., one-half lessof the initial dose. A maintenance regimen can include treating thesubject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of bodyweight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg ofbodyweight per day. The maintenance doses are preferably administered nomore than once every 5, 10, or 30 days. Further, the treatment regimenmay last for a period of time which will vary depending upon the natureof the particular disease, its severity and the overall condition of thepatient. In preferred embodiments the dosage may be delivered no morethan once per day, e.g., no more than once per 24, 36, 48, or morehours, e.g., no more than once every 5 or 8 days. Following treatment,the patient can be monitored for changes in his condition and foralleviation of the symptoms of the disease state. The dosage of thecompound may either be increased in the event the patient does notrespond significantly to current dosage levels, or the dose may bedecreased if an alleviation of the symptoms of the disease state isobserved, if the disease state has been ablated, or if undesiredside-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable. In one embodiment, a pharmaceuticalcomposition includes a plurality of RNA silencing agent species. Inanother embodiment, the RNA silencing agent species has sequences thatare non-overlapping and non-adjacent to another species with respect toa naturally occurring target sequence. In another embodiment, theplurality of RNA silencing agent species is specific for differentnaturally occurring target genes. In another embodiment, the RNAsilencing agent is allele specific. In another embodiment, the pluralityof RNA silencing agent species target two or more SNP alleles (e.g.,two, three, four, five, six, or more SNP alleles).

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the RNA silencing agent composition is an amountsufficient to be effective in treating or preventing a disorder or toregulate a physiological condition in humans. The concentration oramount of RNA silencing agent administered will depend on the parametersdetermined for the agent and the method of administration, e.g. nasal,buccal, or pulmonary. For example, nasal formulations tend to requiremuch lower concentrations of some ingredients in order to avoidirritation or burning of the nasal passages. It is sometimes desirableto dilute an oral formulation up to 10-100 times in order to provide asuitable nasal formulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an RNA silencing agent caninclude a single treatment or, preferably, can include a series oftreatments. It will also be appreciated that the effective dosage of anRNA silencing agent for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result andbecome apparent from the results of diagnostic assays as describedherein. For example, the subject can be monitored after administering anRNA silencing agent composition. Based on information from themonitoring, an additional amount of the RNA silencing agent compositioncan be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target RNA, e.g., an RNA expressed in a neural cell. Thetransgenic animal can be deficient for the corresponding endogenous RNA.In another embodiment, the composition for testing includes an RNAsilencing agent that is complementary, at least in an internal region,to a sequence that is conserved between the target RNA in the animalmodel and the target RNA in a human.

X. Pharmaceutical Compositions and Methods of Administration

The invention pertains to uses of the above-described agents forprophylactic and/or therapeutic treatments as described infra.Accordingly, the modulators (e.g., RNAi agents) of the present inventioncan be incorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the nucleic acidmolecule, protein, antibody, or modulatory compound and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The RNA silencing agents can also be administered by transfection orinfection using methods known in the art, including but not limited tothe methods described in McCaffrey et al. (2002), Nature, 418(6893),38-9 (hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol.,20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J.Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst.Pharm. 53(3), 325 (1996).

The RNA silencing agents can also be administered by any method suitablefor administration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al. (1998), Clin. Immunol.Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat.No. 6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Althoughcompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

As defined herein, a therapeutically effective amount of a RNA silencingagent (i.e., an effective dosage) depends on the RNA silencing agentselected. For instance, if a plasmid encoding shRNA is selected, singledose amounts in the range of approximately 1:g to 1000 mg may beadministered; in some embodiments, 10, 30, 100 or 1000 :g may beadministered. In some embodiments, 1-5 g of the compositions can beadministered. The compositions can be administered one from one or moretimes per day to one or more times per week; including once every otherday. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a protein, polypeptide, or antibodycan include a single treatment or, preferably, can include a series oftreatments.

The nucleic acid molecules of the invention can be inserted intoexpression constructs, e.g., viral vectors, retroviral vectors,expression cassettes, or plasmid viral vectors, e.g., using methodsknown in the art, including but not limited to those described in Xia etal., (2002), supra. Expression constructs can be delivered to a subjectby, for example, inhalation, orally, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91,3054-3057). The pharmaceutical preparation of the delivery vector caninclude the vector in an acceptable diluent, or can comprise a slowrelease matrix in which the delivery vehicle is imbedded. Alternatively,where the complete delivery vector can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can include one or more cells which produce the genedelivery system.

The nucleic acid molecules of the invention can also include smallhairpin RNAs (shRNAs), and expression constructs engineered to expressshRNAs. Transcription of shRNAs is initiated at a polymerase III (polIII) promoter, and is thought to be terminated at position 2 of a4-5-thymine transcription termination site. Upon expression, shRNAs arethought to fold into a stem-loop structure with 3′ UU-overhangs;subsequently, the ends of these shRNAs are processed, converting theshRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp etal. (2002), Science, 296, 550-553; Lee et al, (2002). supra; Miyagishiand Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al.(2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al. (2002),supra.

The expression constructs may be any construct suitable for use in theappropriate expression system and include, but are not limited toretroviral vectors, linear expression cassettes, plasmids and viral orvirally-derived vectors, as known in the art. Such expression constructsmay include one or more inducible promoters, RNA Pol III promotersystems such as U6 snRNA promoters or H1 RNA polymerase III promoters,or other promoters known in the art. The constructs can include one orboth strands of the siRNA. Expression constructs expressing both strandscan also include loop structures linking both strands, or each strandcan be separately transcribed from separate promoters within the sameconstruct. Each strand can also be transcribed from a separateexpression construct, Tuschl (2002), supra.

In certain exemplary embodiments, a composition that includes an RNAsilencing agent of the invention can be delivered to the nervous systemof a subject by a variety of routes. Exemplary routes includeintrathecal, parenchymal (e.g., in the brain), nasal, and oculardelivery. The composition can also be delivered systemically, e.g., byintravenous, subcutaneous or intramuscular injection, which isparticularly useful for delivery of the RNA silencing agents toperipheral neurons. A preferred route of delivery is directly to thebrain, e.g., into the ventricles or the hypothalamus of the brain, orinto the lateral or dorsal areas of the brain. The RNA silencing agentsfor neural cell delivery can be incorporated into pharmaceuticalcompositions suitable for administration. For example, compositions caninclude one or more species of an RNA silencing agent and apharmaceutically acceptable carrier. The pharmaceutical compositions ofthe present invention may be administered in a number of ways dependingupon whether local or systemic treatment is desired and upon the area tobe treated. Administration may be topical (including ophthalmic,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, intrathecal, or intraventricular (e.g.,intracerebroventricular) administration.

The route of delivery can be dependent on the disorder of the patient.For example, a subject diagnosed with HD can be administered an anti-httRNA silencing agent of the invention directly into the brain (e.g., intothe globus pallidus or the corpus striatum of the basal ganglia, andnear the medium spiny neurons of the corpos striatum). In addition to anRNA silencing agent of the invention, a patient can be administered asecond therapy, e.g., a palliative therapy and/or disease-specifictherapy. The secondary therapy can be, for example, symptomatic, (e.g.,for alleviating symptoms), neuroprotective (e.g., for slowing or haltingdisease progression), or restorative (e.g., for reversing the diseaseprocess). For the treatment of HD, for example, symptomatic therapiescan include the drugs haloperidol, carbamazepine, or valproate. Othertherapies can include psychotherapy, physiotherapy, speech therapy,communicative and memory aids, social support services, and dietaryadvice.

An RNA silencing agent can be delivered to neural cells of the brain.Delivery methods that do not require passage of the composition acrossthe blood-brain barrier can be utilized. For example, a pharmaceuticalcomposition containing an RNA silencing agent can be delivered to thepatient by injection directly into the area containing thedisease-affected cells. For example, the pharmaceutical composition canbe delivered by injection directly into the brain. The injection can beby stereotactic injection into a particular region of the brain (e.g.,the substantia nigra, cortex, hippocampus, striatum, or globuspallidus). The RNA silencing agent can be delivered into multipleregions of the central nervous system (e.g., into multiple regions ofthe brain, and/or into the spinal cord). The RNA silencing agent can bedelivered into diffuse regions of the brain (e.g., diffuse delivery tothe cortex of the brain).

In one embodiment, the RNA silencing agent can be delivered by way of acannula or other delivery device having one end implanted in a tissue,e.g., the brain, e.g., the substantia nigra, cortex, hippocampus,striatum or globus pallidus of the brain. The cannula can be connectedto a reservoir of RNA silencing agent. The flow or delivery can bemediated by a pump, e.g., an osmotic pump or minipump, such as an Alzetpump (Durect, Cupertino, Calif.). In one embodiment, a pump andreservoir are implanted in an area distant from the tissue, e.g., in theabdomen, and delivery is effected by a conduit leading from the pump orreservoir to the site of release. Devices for delivery to the brain aredescribed, for example, in U.S. Pat. Nos. 6,093,180, and 5,814,014.

An RNA silencing agent of the invention can be further modified suchthat it is capable of traversing the blood brain barrier. For example,the RNA silencing agent can be conjugated to a molecule that enables theagent to traverse the barrier. Such modified RNA silencing agents can beadministered by any desired method, such as by intraventricular orintramuscular injection, or by pulmonary delivery, for example.

An RNA silencing agent of the invention can be administered ocularly,such as to treat retinal disorder, e.g., a retinopathy. For example, thepharmaceutical compositions can be applied to the surface of the eye ornearby tissue, e.g., the inside of the eyelid. They can be appliedtopically, e.g., by spraying, in drops, as an eyewash, or an ointment.Ointments or droppable liquids may be delivered by ocular deliverysystems known in the art such as applicators or eye droppers. Suchcompositions can include mucomimetics such as hyaluronic acid,chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinylalcohol), preservatives such as sorbic acid, EDTA or benzylchroniumchloride, and the usual quantities of diluents and/or carriers. Thepharmaceutical composition can also be administered to the interior ofthe eye, and can be introduced by a needle or other delivery devicewhich can introduce it to a selected area or structure. The compositioncontaining the RNA silencing agent can also be applied via an ocularpatch.

In general, an RNA silencing agent of the invention can be administeredby any suitable method. As used herein, topical delivery can refer tothe direct application of an RNA silencing agent to any surface of thebody, including the eye, a mucous membrane, surfaces of a body cavity,or to any internal surface. Formulations for topical administration mayinclude transdermal patches, ointments, lotions, creams, gels, drops,sprays, and liquids. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Topical administration can also be used as a means toselectively deliver the RNA silencing agent to the epidermis or dermisof a subject, or to specific strata thereof, or to an underlying tissue.

Compositions for intrathecal or intraventricular (e.g.,intracerebroventricular) administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Compositions for intrathecal or intraventricularadministration preferably do not include a transfection reagent or anadditional lipophilic moiety besides, for example, the lipophilic moietyattached to the RNA silencing agent.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

An RNA silencing agent of the invention can be administered to a subjectby pulmonary delivery. Pulmonary delivery compositions can be deliveredby inhalation of a dispersion so that the composition within thedispersion can reach the lung where it can be readily absorbed throughthe alveolar region directly into blood circulation. Pulmonary deliverycan be effective both for systemic delivery and for localized deliveryto treat diseases of the lungs. In one embodiment, an RNA silencingagent administered by pulmonary delivery has been modified such that itis capable of traversing the blood brain barrier.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An RNAsilencing agent composition may be stably stored as lyophilized orspray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition for inhalation can be mediatedby a dosing timing element which can include a timer, a dose counter,time measuring device, or a time indicator which when incorporated intothe device enables dose tracking, compliance monitoring, and/or dosetriggering to a patient during administration of the aerosol medicament.

The types of pharmaceutical excipients that are useful as carriersinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, trehalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

An RNA silencing agent of the invention can be administered by oral andnasal delivery. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily. In one embodiment, an RNAsilencing agent administered by oral or nasal delivery has been modifiedto be capable of traversing the blood-brain barrier.

In one embodiment, unit doses or measured doses of a composition thatinclude RNA silencing agents are dispensed by an implanted device. Thedevice can include a sensor that monitors a parameter within a subject.For example, the device can include a pump, such as an osmotic pump and,optionally, associated electronics.

An RNA silencing agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

XI. Kits

In certain other aspects, the invention provides kits that include asuitable container containing a pharmaceutical formulation of an RNAsilencing agent, e.g., a double-stranded RNA silencing agent, or sRNAagent, (e.g., a precursor, e.g., a larger RNA silencing agent which canbe processed into a sRNA agent, or a DNA which encodes an RNA silencingagent, e.g., a double-stranded RNA silencing agent, or sRNA agent, orprecursor thereof). In certain embodiments the individual components ofthe pharmaceutical formulation may be provided in one container.Alternatively, it may be desirable to provide the components of thepharmaceutical formulation separately in two or more containers, e.g.,one container for an RNA silencing agent preparation, and at leastanother for a carrier compound. The kit may be packaged in a number ofdifferent configurations such as one or more containers in a single box.The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES

Unlike other types of autosomal dominant diseases, Huntington's diseasedoes not contain a point mutation e.g., a single nucleotide change.Therefore, the strategy to design siRNA directed against a pointmutation in the disease allele cannot be implemented. Instead, thepresent invention directs designed siRNAs against polymorphisms in theHuntingtin gene, of which there are about 30 available in GenBank. Thepresent invention also identifies the polymorphism in the Huntingtondisease allele which differs from the wild type allele, so that siRNAdestroys only the disease mRNA and leaves intact the wild type (normal)allele mRNA. Thus, only the mutant Huntingtin protein is destroyed andthe normal protein is intact.

Example I Testing of RNAi Agents (e.g., siRNAs) Against Mutant Htt inDrosophila Lysates

A siRNA targeting position 2886 in the htt mRNA was designed asdescribed supra. The sequence of the siRNA is depicted in FIG. 5 a (SEQID NO:24 sense; 25 anti-sense). Synthetic RNA (Dharmacon) wasdeprotected according to the manufacturer's protocol. siRNA strands wereannealed (Elbashir et al., 2001a).

Target RNAs were prepared as follows. Target RNAs were transcribed withrecombinant, histidine-tagged, T7 RNA polymerase from PCR products asdescribed (Nykänen et al., 2001; Hutvágner et al., 2002). PCR templatesfor htt sense and anti-sense were generated by amplifying 0.1 ng/ml(final concentration) plasmid template encoding htt cDNA using thefollowing primer pairs: htt sense target, 5′-GCG TAA TAC GAC TCA CTA TAGGAA CAG TAT GTCTCA GAC ATC-3′ (SEQ ID NO:30) and 5′-UUCG AAG UAU UCC GCGUAC GU-3′ (SEQ ID NO:31); htt anti-sense target, 5′-GCG TAA TAC GAC TCACTA TAG GAC AAG CCT AAT TAG TGA TGC-3′ (SEQ ID NO:32) and 5′-GAA CAG TATGTC TCA GAC ATC-3′ (SEQ ID NO:33).

The siRNA was tested using an in vitro RNAi assay, featuring Drosophilaembryo lysates. In vitro RNAi reactions and analysis was carried out aspreviously described (Tuschl et al., 1999; Zamore et al., 2000; Haley etal., 2003). Target RNAs were used at ˜5 nM concentration so thatreactions are mainly under single-turnover conditions. Target cleavageunder these conditions is proportionate to siRNA concentration.

FIG. 5 a shows the efficacy of the siRNA directed against position 2886in the mutant htt. The data clearly demonstrate that the siRNA directscleavage of the sense target to a greater degree than observed for theanti-sense target. However, it is noticed that this first-designed siRNAdid not produce a very active molecule, at least in this in vitro assay.Thermodynamic analysis of the base pair strength at the two ends of thesiRNA duplex indicated roughly equivalent base pair strengths. FIG. 4depicts the thermodynamic analysis of siRNA sense (SEQ ID NO:20; 22respectively) and anti-sense (SEQ ID NO:21; 23 respectively) strand 5′ends for the siRNA duplex in 5a. ΔG (kcal/mole) was calculated in 1MNaCl at 37° C.

To improved the efficacy of the designed siRNA duplex, the 5′ end of thesense strand or position 19 of the anti-sense strand of the htt siRNAtested in FIG. 5 a was altered to produce siRNA duplexes in which the 5′end of the sense strand was either fully unpaired (FIG. 5 c; SEQ ID NO:28 sense; SEQ ID NO:29 anti-sense) or in an A:U base pair (FIG. 5 b; SEQID NO:26 sense; SEQ ID NO:27 anti-sense). The unpairing the 5′ end of ansiRNA strand—the sense strand, in this case—causes that strand tofunction to the exclusion of the other strand. When the htt sense strand5′ end was present in an A:U base pair and the htt anti-sense strand 5′end was in a G:C pair, the sense strand dominated the reaction (FIG. 5b-c), but the htt anti-sense strand retained activity similar to thatseen for the originally-designed siRNA.

Example II RNAi Knockdown of Htt Protein in Cultured Cells

In a first experiment, siRNAs targeting a polymorphism in the htt mRNA(i.e., the polymorphism at position 2886 in the htt mRNA) were testedfor their ability to down-regulate endogenous Htt protein in HeLa cells.HeLa cells were cultures and transfected as follows. HeLa cells weremaintained at 37° C. in Dulbecco's modified Eagle's medium (DMEM,Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 unit/mlpenicillin and 100 μg/ml streptomycin (Invitrogen). Cells were regularlypassaged at sub-confluence and plated at 70% confluency 16 hours beforetransfection. Lipofectamine™ (Invitrogen)-mediated transienttransfection of siRNAs were performed in duplicate 6-well plates(Falcon) as described for adherent cell lines by the manufacturer. Astandard transfection mixture containing 100-150 nM siRNA and 9-10 μlLipofectamine™ in 1 ml serum-reduced OPTI-MEM® (Invitrogen) was added toeach well. Cells were incubated in transfection mixture at 37 C for 6hours and further cultured in antibiotic-free DMEM. For Western blotanalysis at various time intervals, the transfected cells wereharvested, washed twice with phosphate buffered saline (PBS,Invitrogen), flash frozen in liquid nitrogen, and stored at −80° C. foranalysis.

Three siRNAs were tested against a common target sequence in exon 1 andfour siRNAs were tested for the position 2886 polymorphism. Western blotanalysis was performed as follows. Cells treated with siRNA wereharvested as described above and lysed in ice-cold reporter lysis buffer(Promega) containing protease inhibitor (complete, EDTA-free, 1tablet/10 ml buffer, Roche Molecular Biochemicals). After clearing theresulting lysates by centrifugation, protein in clear lysates wasquantified by Dc protein assay kit (Bio-Rad). Proteins in 60 μg of totalcell lysate were resolved by 10% SDS-PAGE, transferred onto apolyvinylidene difluoride membrane (PVDF, Bio-Rad), and immuno-blottedwith antibodies against CD80 (Santa Cruz). Protein content wasvisualized with a BM Chemiluminescence Blotting Kit (Roche MolecularBiochemicals). The blots were exposed to x-ray film (Kodak MR-1) forvarious times (30 s to 5 min). FIG. 6 a depicts the results of theWestern analysis. Tubulin served as the loading control. The data arequantified and normalized in FIG. 6 b. Of the siRNAs tested, 2886-4,reproducibly showed enhanced efficacy in cultured HeLa cells (FIG. 6).This siRNA also reproducibly showed enhanced efficacy in vitro (notshown). GFP siRNA is a control siRNA that shares no sequence homologywith htt mRNA.

siRNAs against polymorphic regions in the htt mRNA can likewise betested in cells transfected with human htt cDNA or in cells transfectedwith htt reporter constructs. Lipofectamine™ (Invitrogen)-mediatedtransient cotransfections of cDNAs or reporter plasmids and siRNAs areperformed as described supra. To test the ability of siRNAs to targethtt reported constructs, RNAi was used to inhibit GFP-htt expression incultured human Hela cell lines. Briefly, HeLa cells were transfectedwith GFP-htt siRNA duplex, targeting the GFP-htt mRNA sequence. Toanalyze RNAi effects against GFP-htt, lysates were prepared from siRNAduplex-treated cells at various times after transfection. Western blotexperiments were carried out as described supra. Briefly, HeLa cellswere harvested at various times post transfection, their protein contentwas resolved on 10% SDS-PAGE, transferred onto PVDF membranes, andimmunoblotted with appropriate antibodies. Results of this studyindicated that siRNA against GFP can eliminate expression of GFP-httexpression in Hela cells transfected with the GFP-htt gene. For studiestargeting exogenously introduces htt, procedures are as described exceptthat anti-Htt antibodies are used for immunoblotting.

RNAi can be used to inhibit htt expression in cultured neuronal cells aswell. Exemplary cells include PC12 (Scheitzer et al., Thompson et al.)and NT3293 (Tagle et al.) cell lines as previously described. Additionalexemplary cells include stably-transfected cells, e.g. neuronal cells orneuronally-derived cells. PC12 cell lines expressing exon 1 of the humanhuntingtin gene (Htt) can be used although expression of exon 1 reducescell survival. GFP-Htt PC12 cells having an inducible GFP-Htt gene canalso be used to test or validate siRNA efficacy.

Example III Htt siRNA Delivery in an In Vivo Setting

R6/2 mice models (expressing the R6/2 human htt cDNA product) are anaccepted animal model to study the effectiveness of siRNA delivery in anin vivo setting. Genetically engineered R6/2 mice were used to test theeffectiveness of siRNA at the 5′ terminus of huntingtin mRNA. Htt siRNAwas injected into the striatum of R6/2 mice through an Alzet pump. Micewere treated for 14 days with the siRNA/Alzet pump delivery system.

Results of this study indicated that two mice receiving the siRNA withTrans-IT TKO (Minis) as either a 20 or 200 nM solution at 0.250/hourshowed no deterioration of motor impairment from day 67 to day 74.Generally, these R6/2 are expected to have a continued reduction inrotarod beyond day 60.

Example IV SNP Analysis in Patients with Huntington's Disease (HD)

Single nucleotide polymorphism (SNP) sequencing analysis was performedto identify heterozygotic SNPs in patients with Huntington's Disease(HD). DNA samples were obtained from brain repositories in Charlestown,Mass., USA and New York City, N.Y. and a DNA repository in Ulm, Germany.

In one study, a total of 195 subjects (HD and control) were examined.Twenty-one SNP sites were identified; eighteen were reported in SNPPERand three have not previously been reported. In this population, fourreported SNP sites were not confirmed.

149/195 subjects (76%) contained SNP heterozygosity. The USA and Germanpopulations had 76% heterozygosity at SNP sites. HD patients andcontrols had the same frequency of SNP heterozygosity. Six SNP sites hadan allelic frequency >30%.

In a second study, a total of twelve candidate polymorphic sites weresequenced in more than 107 HD brain specimens. Fifty-five percent of HDpatients in the USA specimens (26/47; USA) and 57% of controls (50/88;USA) were heterozygous at one or more SNP site. Thirty-eight percent ofHD patients in the German collection (23/60; Germany) were heterozygousat one or more polymorphic site. Forty-three of the total 107 HDpatients had clusters of heterozygous polymorphic sites, withheterozygosity at five or eight SNP sites.

Example V Testing of RNA Silencing Agents (e.g., siRNAs) Against HttSNPs

Two SNPs (at genomic positions RS 363125 and RS 362331) were selected totest RNAi discrimination in an in vitro reporter assay.

SiRNAs targeting each SNP were designed as described supra. siRNAstrands were annealed (Elbashir et al., 2001a).

Target RNAs may be prepared as follows. Target RNAs are transcribed withrecombinant, histidine-tagged, T7 RNA polymerase from PCR products asdescribed (Nykänen et al., 2001; Hutvagner et al., 2002). PCR templatesfor htt sense and anti-sense target RNA preparation are generated byamplifying plasmid template encoding htt cDNA using primer pairs. Eachprimer pair consists of forward and reverse primers which arecomplementary to regions that are immediately upstream and downstream,respectively, of one of the two alleles of a heterozygous SNP site.

Each siRNA was tested using a cell-based in vitro luciferase reporterassay. In vitro RNAi reactions and analysis may also be carried out inDrosophila embryo lysates as previously described (Tuschl et al., 1999;Zamore et al., 2000; Haley et al., 2003). Target RNAs are used at ˜5 nMconcentration so that reactions are mainly under single-turnoverconditions. Target cleavage under these conditions is proportionate tosiRNA concentration.

In RS363125 (predicted high discriminator), 80% knockdown was achievedat 0.5 nM siRNA for a siRNA having a guide strand perfectlycomplementary to a target mRNA containing the polymorphic adenosine(“A”) SNP allele of the target SNP site from huntingtin, but containinga mismatch with a non-target mRNA containing the corresponding cytosine(“C”) SNP allele of the heterozygous RS363125 SNP site (FIG. 10A). Asingle nucleotide mismatch effectively discriminated between bothalleles of this SNP as the siRNA did not achieve the save level ofknockdown of non-target (“mismatched”) mRNA, even at up to 20 nM siRNA(FIG. 10B). In contrast, an siRNA having perfect complementary to the“C” SNP allele and a mismatch with the “A” SNP allele of the RS363125SNP site (FIG. 11A) was effective in achieving discriminatory RNAi infavor of the “C” SNP allele (FIG. 11B).

Similar findings applied to the RS362331 polymorphism. The sequences ofthe 21-mer siRNAs targeting each SNP allele (“C” or “U”) of the RS362331polymorphism are depicted in FIG. 8A (SEQ ID NO: 34 sense; SEQ ID NO: 35anti-sense) and FIG. 9A (SEQ ID NO: 38 sense; SEQ ID NO: 39 anti-sense).Each siRNA is perfectly complementary to the targeted SNP allele, butcontains a mismatch with the non-targeted SNP allele at position 10 ofthe guide strand (P10).

FIG. 8B shows the efficacy of a siRNA directed against the SNP allele ofRS362331 having a cytosine (“C”) at the heterozygotic SNP site (SEQ IDNO:36; match). The data clearly demonstrate that the siRNA directscleavage of the matched target SNP allele to a greater degree thanobserved for the mismatched target SNP allele (SEQ ID NO:37; mismatch).Similarly, FIG. 9B, shows the efficacy of siRNA against the SNP alleleof RS362331 which has a uridine (“U”) at the heterozygotic SNP site (SEQID NO: 37; match). Greater than 50% knockdown of the targeted SNP alleleis achieved at 0.5 nM siRNA, while the non-targeted “C” SNP allele (SEQID NO:36; mismatch) is relatively unaffected.

The efficacy of siRNA directed against the homozygous C allele (SEQ IDNO: 48) in the 3′UTR of Htt gene was also tested. siRNAs having a guidestrand with perfect complementary to the C allele (FIG. 12A) orcontaining a single U:C mismatch with the C allele at position 10 (P10)of the guide strand (FIG. 12B) were designed. Transfection was performedat an siRNA concentration of 20 nM in HEK cells homozygous for the Callele. Transfection efficiency was approximately 70%. As depicted inFIG. 12C and FIG. 12D, matched siRNAs were much more effective thanmismatched siRNAs in achieving knockdown of both Htt target mRNA andprotein levels, respectively. This pattern of gene silencing wasconsistently observed at lower siRNA concentrations (5 nM and 10 nM) aswell (see FIG. 13).

These results indicate that siRNAs can preferentially silence one of thetwo SNP alleles differing at the polymorphic site and that heterozygousSNP sites in huntingtin are attractive targets for therapeutic siRNAs.

Example VI Effect of RNA Silencing Agents (e.g., siRNAs) on Mutant HttProtein Expression

Western blotting may also be employed to test the ability of siRNAs todown-regulate endogenous Htt protein in cultured cells using any of thetechniques described infra.

RNA silencing activity of SNP-specific siRNAs can be tested HeLa cells.HeLa cells are maintained at 37° C. in Dulbecco's modified Eagle'smedium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum(FBS), 100 unit/ml penicillin and 100 μg/ml streptomycin (Invitrogen).Cells are regularly passaged at sub-confluence and plated at 70%confluency 16 hours before transfection. Lipofectamine™(Invitrogen)-mediated transient transfection of siRNAs are performed induplicate 6-well plates (Falcon) as described for adherent cell lines bythe manufacturer. A standard transfection mixture containing 100-150 nMsiRNA and 9-10 μl Lipofectamine™ in 1 ml serum-reduced OPTI-MEM®(Invitrogen) are added to each well. Cells are incubated in transfectionmixture at 37 C for 6 hours and further cultured in antibiotic-freeDMEM. For Western blot analysis at various time intervals, thetransfected cells are harvested, washed twice with phosphate bufferedsaline (PBS, Invitrogen), flash frozen in liquid nitrogen, and stored at−80° C. for analysis.

Western blot analysis is performed as follows. Cells treated with siRNAare harvested as described above and lysed in ice-cold reporter lysisbuffer (Promega) containing protease inhibitor (complete, EDTA-free, 1tablet/10 ml buffer, Roche Molecular Biochemicals). After clearing theresulting lysates by centrifugation, protein in clear lysates isquantified by Dc protein assay kit (Bio-Rad). Proteins in 60 pg of totalcell lysate are resolved by 10% SDS-PAGE, transferred onto apolyvinylidene difluoride membrane (PVDF, Bio-Rad), and immuno-blottedwith antibodies against CD80 (Santa Cruz). Protein content viasvisualized with a BM Chemiluminescence Blotting Kit (Roche MolecularBiochemicals). The blots are exposed to x-ray film (Kodak MR-1) forvarious times (30 s to 5 min).

siRNAs against polymorphic regions in the htt mRNA can likewise betested in cells transfected with human htt cDNA or in cells transfectedwith htt reporter constructs. Lipofectamine™ (Invitrogen)-mediatedtransient cotransfections of cDNAs or reporter plasmids and siRNAs areperformed as described supra. To test the ability of siRNAs to targethtt reported constructs, RNAi is used to inhibit GFP-htt expression incultured human Hela cell lines. Briefly, HeLa cells are transfected withGFP-htt siRNA duplex, targeting the GFP-htt mRNA sequence. To analyzeRNAi effects against GFP-htt, lysates are prepared from siRNAduplex-treated cells at various times after transfection. Western blotexperiments are carried out as described supra.

Inhibition of htt expression can be assessed in cultured neuronal cellsas well. Exemplary cells include PC12 (Scheitzer et al., Thompson etal.) and NT3293 (Tagle et al.) cell lines as previously described.Additional exemplary cells include stably-transfected cells, e.g.neuronal cells or neuronally-derived cells. PC12 cell lines expressingexon 1 of the human huntingtin gene (Htt) can be used althoughexpression of exon 1 reduces cell survival. GFP-Htt PC12 cells having aninducible GFP-Htt gene can also be used to test or validate siRNAefficacy.

Example VII Htt siRNA Delivery in an In Vivo Setting

R6/2 mice models (expressing the R6/2 human htt cDNA product) are anaccepted animal model to study the effectiveness of siRNA delivery in anin vivo setting. Genetically engineered R6/2 mice may be used to testthe effectiveness of siRNA at the 5′ terminus of huntingtin mRNA. HttsiRNA are injected into the striatum of R6/2 mice through an Alzet pump.Mice are treated for 14 days with the siRNA/Alzet pump delivery system.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

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Zeng et al., (2002)

1. A method of treating a subject having or at risk for a diseasecharacterized or caused by a gain-of-function mutant protein,comprising: administering to said subject an effective amount of a RNAiagent targeting an allelic polymorphism within a gene encoding saidmutant protein, such that sequence-specific interference of said geneoccurs; thereby treating said disease in said subject.
 2. The method ofclaim 1, wherein said gene comprises an expanded trinucleotide repeatregion.
 3. The method of claim 1, wherein said mutant protein comprisesan expanded polyglutamine domain.
 4. The method of claim 1, wherein thedisease is selected from the group consisting of Huntington's disease,spino-cerebellar ataxia type 1, spino-cerebellar ataxia type 2,spino-cerebellar ataxia type 3, spino-cerebellar ataxia type 6,spino-cerebellar ataxia type 7, spino-cerebellar ataxia type 8,spino-cerebellar ataxia type 12, fragile X syndrome, fragile XE MR,Friedreich ataxia, myotonic dystrophy, spinal bulbar muscular diseaseand dentatoiubral-pallidoluysian atrophy.
 5. The method of claim 4,wherein the disease is Huntington's disease.
 6. The method of claim 5,wherein the RNAi agent targets an allelic polymorphism within the geneencoding a huntingtin protein.
 7. The method of claim 5, wherein theRNAi agent targets a polymorphism selected from the group consisting ofP1-P5.
 8. The method of claim 5, wherein the RNAi agent targets apolymorphism selected from the group consisting of P6-P43.
 9. The methodof claim 1, wherein the RNAi agent comprises a first strand comprisingabout 16-25 nucleotides homologous to a region of the gene comprisingthe polymorphism and a second strand comprising about 16-25 nucleotidescomplementary to the first strand.
 10. The method of claim 1, whereinthe effective amount is an amount effective to inhibit the expression oractivity of the mutant protein.
 11. An RNAi agent comprising a firststrand comprising about 16-25 nucleotides homologous to a region of agene encoding a gain-of-function mutant protein, said region comprisingan allelic polymorphism, and a second strand comprising about 16-25nucleotides complementary to the first strand, wherein the RNAi agentdirect target-specific cleavage of a mRNA transcribed from the geneencoding the mutant protein.
 12. The RNAi agent of claim 11, whichtargets a polymorphism within the gene encoding a Huntington protein.13. The RNAi agent of claim 12, wherein said polymorphism is selectedfrom the group comprising P1-P5.
 14. The RNAi agent of claim 12, whereinsaid polymorphism is selected from the group comprising P6-P43.
 15. TheRNAi agent of claim 11, wherein the first strand comprises a nucleotidesequence identical to the sequence of the polymorphism.
 16. The RNAiagent of claim 11, further comprising a loop portion comprising 4-11nucleotides that connects the two strands.
 17. An isolated nucleic acidmolecule encoding the RNAi agent of claim
 11. 18. A vector comprisingthe nucleic acid molecule of claim
 17. 19. The vector of claim 18, whichis a viral vector, retroviral vector, expression cassette, or plasmid.20. The vector of claim 18, further comprising an RNA Polymerase III orRNA Polymerase II promoter.
 21. The vector of claim 18, wherein the RNAPolymerase III promoter is the U6 or H1 promoter.
 22. A host cellcomprising the RNAi agent of claim.
 23. A host cell comprising thevector of claim
 18. 24. The host cell of claim 22, which is a mammalianhost cell.
 25. The host cell of claim 24, which is a non-human mammaliancell.
 26. The host cell of claim 24, which is a human cell.
 27. Acomposition comprising the RNAi agent of claim 11, and apharmaceutically acceptable carrier.
 28. A method for treating a diseaseor disorder in a subject caused by a gain-of function mutant protein,comprising identifying an allelic polymorphism within a gene encodingsaid mutant protein and administering to said subject an RNAi agenttargeting said polymorphism such that the mutant protein is decreased,thereby treating the subject.
 29. A method of silencing a target mRNAencoding a mutant huntingtin (htt) protein in a cell, comprisingcontacting the cell with effective amount of a RNA silencing agenttargeting a heterozygous single nucleotide polymorphism (SNP) within thetarget mRNA, such that RNA silencing of said mRNA occurs, wherein theSNP has an allelic frequency of at least 35% in a sample population. 30.The method of claim 29, wherein the SNP is present at genomic siteRS362331.
 31. The method of claim 29, wherein the target mRNA comprisesthe sequence set forth as SEQ ID NO:
 36. 32. The method of claim 29,wherein the target mRNA comprises the sequence set forth as SEQ ID NO:37.
 33. The method of claim 29, wherein the RNA silencing agent iscapable of inducing discriminatory RNA silencing.
 34. The method ofclaim 29, wherein the antisense strand of said RNA silencing agent iscomplementary to the SNP and wherein said RNA silencing agent is capableof substantially silencing the mutant huntingtin protein withoutsubstantially silencing the corresponding wild-type huntingtin protein.35. The method of claim 29, wherein the sample population is of WesternEuropean origin.
 36. A method of silencing a target mRNA encoding amutant huntingtin (htt) protein in a cell, comprising contacting thecell with an effective amount of a siRNA targeting a heterozygous singlenucleotide polymorphism (SNP) within the target mRNA, such that RNAsilencing of said mRNA occurs, wherein the siRNA is selected from thegroup consisting of: a. an siRNA comprising (i) a sense strandcomprising the sequence set forth as SEQ ID NO: 34 or a variant thereof;and (ii) an antisense strand comprising the sequence set forth as SEQ IDNO: 35 or a variant thereof, said variant comprising at least onenucleotide analog or backbone modification; b. an siRNA comprising (i) asense strand comprising the sequence set forth as SEQ ID NO: 38 or avariant thereof; and (ii) an antisense strand comprising the sequenceset forth as SEQ ID NO: 39 or a variant thereof, said variant comprisingat least one nucleotide analog or backbone modification, c. an siRNAcomprising (i) a sense strand comprising the sequence set forth as SEQID NO: 40 or a variant thereof; and (ii) an antisense strand comprisingthe sequence set forth as SEQ ID NO: 41 or a variant thereof, saidvariant comprising at least one nucleotide analog or backbonemodification, and d. an siRNA comprising (i) a sense strand comprisingthe sequence set forth as SEQ ID NO: 44 or a variant thereof; and (ii)an antisense strand comprising the sequence set forth as SEQ ID NO: 45or a variant thereof, said variant comprising at least one nucleotideanalog or backbone modification.
 37. The method of claim 36, wherein thesiRNA comprises a lipophilic moiety.
 38. The method of claim 37, whereinthe lipophilic moiety is a cholesterol moiety.
 39. An RNA silencingagent comprising an antisense strand comprising about 16-25 nucleotideshomologous to a region of an mRNA encoding a mutant huntingtin (htt)protein, said region comprising a heterozygous single nucleotidepolymorphism (SNP) having an allelic frequency of at least 35% in asample population, wherein the RNA silencing agent is capable ofdirecting RNA silencing of said mRNA.
 40. The RNA silencing agent ofclaim 39, wherein the SNP is present at genomic site RS362331.
 41. TheRNA silencing agent of claim 39, wherein the target mRNA comprises thesequence set forth as SEQ ID NO:
 36. 42. The RNA silencing agent ofclaim 39, wherein the target mRNA comprises the sequence set forth asSEQ ID NO:
 37. 43. The RNA silencing agent of claim 39, which is capableof inducing discriminatory RNA silencing.
 44. The RNA silencing agent ofclaim 39, which is capable of substantially silencing the mutanthuntingtin protein without substantially silencing the correspondingwild-type huntingtin protein.
 45. The RNA silencing agent of claim 39,wherein the RNA silencing agent is an siRNA.
 46. The siRNA of claim 45,comprising a sense strand having a nucleotide sequence identical to thesequence of the SNP.
 47. The siRNA of claim 39, wherein the sense strandof the siRNA is identical to the polymorphism at a nucleotide positionthat is 10 nucleotides from the 5′ end of the antisense strand.
 48. ThesiRNA of claim 39, wherein the sense strand of the siRNA is identical tothe polymorphism at a nucleotide position that is 16 nucleotides fromthe 5′ end of the antisense strand.
 49. An siRNA selected from the groupconsisting of: a. an siRNA comprising (i) a sense strand comprising thesequence set forth as SEQ ID NO: 34 or a variant thereof; and (ii) anantisense strand comprising the sequence set forth as SEQ ID NO: 35 or avariant thereof, said variant comprising at least one nucleotide analogor backbone modification; b. an siRNA comprising (i) a sense strandcomprising the sequence set forth as SEQ ID NO: 38 or a variant thereof;and (ii) an antisense strand comprising the sequence set forth as SEQ IDNO: 39 or a variant thereof, said variant comprising at least onenucleotide analog or backbone modification, c. an siRNA comprising (i) asense strand comprising the sequence set forth as SEQ ID NO: 40 or avariant thereof; and (ii) an antisense strand comprising the sequenceset forth as SEQ ID NO: 41 or a variant thereof, said variant comprisingat least one nucleotide analog or backbone modification, and d. an siRNAcomprising (i) a sense strand comprising the sequence set forth as SEQID NO: 44 or a variant thereof; and (ii) an antisense strand comprisingthe sequence set forth as SEQ ID NO: 45 or a variant thereof, saidvariant comprising at least one nucleotide analog or backbonemodification.
 50. The siRNA of claim 49, wherein the siRNA comprises alipophilic moiety.
 51. The siRNA of claim 50, wherein the lipophilicmoiety is a cholesterol moiety.